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  • 101.
    Ellencrona, Karin
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Functional characterization of interactions between the flavivirus NS5 protein and PDZ proteins of the mammalian host2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Flaviviruses are found all over the world and affect and infect millions of people every year. Flavivirus infection can lead to severe clinical outcomes resulting in neuronal damages e.g. Tick-borne encephalitis virus (TBEV), or severe hemorrhagic fevers e.g. Dengue virus (DENV). In order to effectively treat infected patients and to prevent these diseases we must understand how these viruses work and how they interfere with the mammalian host. This thesis is focusing on interactions between the virus protein NS5 and human host cell proteins. The interactions presented here might be key factors for out-come of viral disease. NS5 is the largest of the non-structural proteins and is essential for the replication and the capping as it contains both RNA dependent RNA polymerase and Methyltransferase domains. We found that TBEV NS5 interacts with human PDZ domain protein Scribble, a polarization protein important e.g. in regulating membrane trafficking. We determined that the interaction depend on a novel internal motif in TBEVNS5. This interaction could be correlated to NS5s ability to interfere with the immune system as absence of Scribble prevented NS5 from blocking phosphorylation of STAT upon Interferon induction. The role of NS5 in human PDZ domain targeting was addressed further by using a PDZ array system. Both TBEVNS5 and DENVNS5 bind additional PDZ domains using the internal motif. The tight junction protein ZO-1 binds both DENVNS5 and TBEVNS5. DENVNS5 is mainly present in the nucleus and co-localize with ZO-1 in un-polarized cells. In polarized cells TBEVNS5 and ZO-1 co-localize at the plasmamembrane. Putative C-terminal PDZ binding motifs of TBEVNS5 and WNVNS5 were characterized using the PDZ array system. This detected four novel binding partners of TBEVNS5 but numerous of potential WNVNS5 binding partners. We found that TBEVNS5 co-localizes with ZO-2 in the cellular membrane. Further, we found that TBEVNS5 induce the AP-1 by a 2 fold over the control.

  • 102.
    Ellencrona, Karin
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Syed, Asim
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Johansson, Magnus
    Södertörns högskola.
    Flavivirus NS5 associates with host-cell proteins zonula occludens-1 (ZO-1) and regulating synaptic membrane exocytosis-2 (RIMS2) via an internal PDZ binding mechanism2009In: Biological chemistry (Print), ISSN 1431-6730, E-ISSN 1437-4315, Vol. 390, p. 319-323Article in journal (Refereed)
    Abstract [en]

    Dengue virus (DENV) and tick-borne encephalitis virus (TBEV) are flaviviruses, which can cause lethal hemorrhagic fever and encephalitis, respectively. Here, we demonstrate that the TBEV-NS5 and DENV-NS5 proteins use an internal binding mechanism to target human PDZ proteins. TBEV-NS5 has high affinity to regulating synaptic membrane exocytosis-2 (RIMS2) and Scribble, whereas DENV-NS5 binds primarily to the tight junction protein zonula occludens-1 (ZO-1). Targeting of TBEVNS5 to the plasma membrane is stabilised by ZO-1; however, DENV-NS5 co-localises with ZO-1 in the nucleus. These interactions have potential important roles in the ability of flaviviruses to manipulate cell proliferation, junction permeability and the interferon pathways.

  • 103.
    Elvers, Ingegerd
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Replication Fork Stability in Mammalian Cells2011Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Maintaining replication fork integrity is vital to preserve genomic stability and avoid cancer. Physical DNA damage and altered nucleotide or protein pools represent replication obstacles, generating replicative stress. Numerous cellular responses have evolved to ensure faithful DNA replication despite such challenges. Understanding those responses is essential to understand and prevent or treat replication-associated diseases, such as cancer.

    Re-priming is a mechanism to allow resumption of DNA synthesis past a fork-stalling lesion. This was recently suggested in yeast and explains the formation of gaps during DNA replication on damaged DNA. Using a combination of assays, we indicate the existence of re-priming also in human cells following UV irradiation.

    The gap left behind a re-primed fork must be stabilised to avoid replication-associated collapse. Our results show that the checkpoint signalling protein CHK1 is dispensable for stabilisation of replication forks after UV irradiation, despite its role in replication fork progression on UV-damaged DNA. It is not known what proteins are necessary for collapse of an unsealed gap or a stalled fork. We exclude one, previously suggested, endonuclease from this mechanism in UV-irradiated human fibroblasts. We also show that focus formation of repair protein RAD51 is not necessarily associated with cellular sensitivity to agents inducing replicative stress, in rad51d CHO mutant cells.

    Multiple factors are required for replication fork stability, also under unperturbed conditions. We identify the histone methyltransferase SET8 as an important player in the maintenance of replication fork stability. SET8 is required for replication fork progression, and depletion of SET8 led to the formation of replication-associated DNA damage.

    In summary, our results increase the knowledge about mechanisms and signalling at replication forks in unperturbed cells and after induction of replicative stress.

  • 104.
    Elvers, Ingegerd
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Replication fork stability in mammalian cells2009Licentiate thesis, monograph (Other academic)
  • 105.
    Elvers, Ingegerd
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Deperas-Kaminska, Marta
    Joint Institute for Nuclear Research, Dubna, Russia.
    Johansson, Fredrik
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Schultz, Niklas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Wojcik, Andrzej
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Helleday, Thomas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    UV-induced replication fork collapse in DNA polymerase η deficient cells is independent of the MUS81 endonucleaseManuscript (preprint) (Other academic)
    Abstract [en]

    The MUS81 endonuclease was initially identified in resonse to UV and MMS lesions, and has been implicated in replication fork collapse after exposure to cross-linking agents. After stalling of replication forks by hydroxyurea treatment, the forks collapse independently of MUS81 but the endonuclease is required for replication fork restart. However in cells deficient in the Werner helicase, MUS81 is needed for collapse of replication forks after hydroxyurea treatment, indicating that the endonuclease might play a role in replication fork collapse in cells with impaired replication. UV irradiation induces DNA damage that physically block replication fork elongation but may be bypassed by translesion synthesis polymerases. Here we have investigated the role of MUS81 after UV irradiation of human fibroblasts deficient in Polη, and restored (wild-type) cells. We show that in wild-type cells, depletion of MUS81 does not affect survival after UV irradiation. However in Polη deficient cells, MUS81 depletion further lowers the survival after exposure to UV. In spite of this, replication forks collapse in UV irradiated Polη deficient cells independently of MUS81.

  • 106.
    Elvers, Ingegerd
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Hagenkort, Anna
    Stockholm University, Science for Life Laboratory (SciLifeLab).
    Johansson, Fredrik
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Djureinovic, Tatjana
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Lagerqvist, Anne
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Schultz, Niklas
    Stockholm University, Science for Life Laboratory (SciLifeLab).
    Stoimenov, Ivaylo
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Erixon, Klaus
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Helleday, Thomas
    Stockholm University, Science for Life Laboratory (SciLifeLab).
    CHK1 activity is required for continuous replication fork elongation but not stabilization of post-replicative gaps after UV irradiation2012In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 40, no 17, p. 8440-8448Article in journal (Refereed)
    Abstract [en]

    Ultraviolet (UV)-induced DNA damage causes an efficient block of elongating replication forks. The checkpoint kinase, CHK1 has been shown to stabilize replication forks following hydroxyurea treatment. Therefore, we wanted to test if the increased UV sensitivity caused by the unspecific kinase inhibitor caffeine-inhibiting ATM and ATR amongst other kinases-is explained by inability to activate the CHK1 kinase to stabilize replicative structures. For this, we used cells deficient in polymerase eta (Pol eta), a translesion synthesis polymerase capable of properly bypassing the UV-induced cis-syn TT pyrimidine dimer, which blocks replication. These cells accumulate gaps behind progressing replication forks after UV exposure. We demonstrate that both caffeine and CHK1 inhibition, equally retards continuous replication fork elongation after UV treatment. Interestingly, we found more pronounced UV-sensitization by caffeine than with the CHK1 inhibitor in clonogenic survival experiments. Furthermore, we demonstrate an increased collapse of replicative structures after caffeine treatment, but not after CHK1 inhibition, in UV-irradiated cells. This demonstrates that CHK1 activity is not required for stabilization of gaps induced during replication of UV-damaged DNA. These data suggest that elongation and stabilization of replicative structures at UV-induced DNA damage are distinct mechanisms, and that CHK1 is only involved in replication elongation.

  • 107.
    Elvers, Ingegerd
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Johansson, Fredrik
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Djureinovic, Tatjana
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Lagerqvist, Anne
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Stoimenov, Ivaylo
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Erixon, Klaus
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Helleday, Thomas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    CHK1 activity is required for replication fork elongation but not stabilisation after UV irradiationArticle in journal (Refereed)
    Abstract [en]

    UV-induced DNA damage cause an efficient block for elongating replication forks. Since CHK1 has been shown to stabilise replication forks following hydroxyurea treatment, we wanted to test if the increased killing with the unspecific kinase inhibitor caffeine, inhibiting ATM and ATR amongst other kinases, is explained by inability to activate the CHK1 kinase to stabilise UV-stalled replication forks. For this, we used cells deficient in Polη, a translesion synthesis polymerase capable of properly bypassing the UV-induced cis-syn TT pyrimidine dimer, which blocks replication. These cells, derived from the variant type of xeroderma pigmentosum, are sensitised to UV irradiation by caffeine treatment. We demonstrate that both caffeine and CHK1 inhibition, using CEP-3891, equally retards replication fork elongation after UV treatment in Polη deficient cells. Interestingly, we found more pronounced UV-sensitisation by caffeine than with the CHK1 inhibitor in clonogenic survival experiments. Furthermore, we demonstrate an increased collapse of UV-stalled forks after caffeine treatment, but not after CHK1 inhibition, demonstrating that CHK1 activity is not required for stabilisation of UV-stalled replication forks. These data suggest that stabilisation and elongation at UV-stalled forks are distinct mechanisms, and that CHK1 is only involved in fork elongation. 

  • 108.
    Elvers, Ingegerd
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Johansson, Fredrik
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Djureinovic, Tatjana
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Lagerqvist, Anne
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Stoimenov, Ivaylo
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Schultz, Niklas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Erixon, Klaus
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Helleday, Thomas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    UVC–stalled replication forks readily collapse into DNA double-strand breaksin the absence of DNA polymerase η and independently of Mus81 in humancellsManuscript (preprint) (Other academic)
  • 109.
    Elvers, Ingegerd
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Johansson, Fredrik
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Groth, Petra
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Erixon, Klaus
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Helleday, Thomas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    UV stalled replication forks restart by re-priming in human fibroblasts2011In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 39, no 16, p. 7049-7057Article in journal (Refereed)
    Abstract [en]

    Restarting stalled replication forks is vital to avoid fatal replication errors. Previously, it was demonstrated that hydroxyurea-stalled replication forks use an active restart mechanism or rescue replication by new origin firing. Using the DNA fiber assay, we find to our surprise no evidence that UV-damaged replication forks are arrested and only detect a slightly reduced fork speed on a UV-damaged template. Interestingly, no evidence for UV-induced fork stalling was observed even in translesion synthesis defective, Polηmut cells. In contrast, using an assay to measure DNA molecule elongation at the fork, we observe that DNA elongation is severely blocked, particularly in UV-damaged Polηmut cells. In conclusion, these data suggest that UV-blocked replication forks restart effectively through re-priming. If left unfilled, the gap behind a re-primed fork may collapse into a DNA double-strand break that is repaired by a recombination pathway, similar to the fate of replication forks collapsed after hydroxyurea treatment.

  • 110. Elväng, Annelie
    et al.
    Melik, Wessam
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Bertrand, Yann
    Lönn, Mikael
    Johansson, Magnus
    Sequencing of a Tick-Borne Encephalitis Virus from Ixodes ricinus Reveals a Thermosensitive RNA Switch Significant for Virus Propagation in Ectothermic Arthropods2011In: Vector Borne and Zoonotic Diseases, ISSN 1530-3667, E-ISSN 1557-7759, Vol. 11, no 6, p. 649-658Article in journal (Refereed)
    Abstract [en]

    Tick-borne encephalitis virus (TBEV) is a flavivirus with major impact on global health. The geographical TBEV distribution is expanding, thus making it pivotal to further characterize the natural virus populations. In this study, we completed the earlier partial sequencing of a TBEV pulled out of a pool of RNA extracted from 115 ticks collected on Torö in the Stockholm archipelago. The total RNA was sufficient for all sequencing of a TBEV genome (Torö-2003), without conventional enrichment procedures such as cell culturing or suckling mice amplification. To our knowledge, this is the first time that the genome of TBEV has been sequenced directly from an arthropod reservoir. The Torö-2003 sequence has been characterized and compared with other TBE viruses. In silico analyses of secondary RNA structures formed by the two untranslated regions revealed a temperature-sensitive structural shift between a closed replicative form and an open AUG accessible form, analogous to a recently described bacterial thermoswitch. Additionally, novel phylogenetic conserved structures were identified in the variable part of the 3′-untranslated region, and their sequence and structure similarity when compared with earlier identified structures suggests an enhancing function on virus replication and translation. We propose that the thermo-switch mechanism may explain the low TBEV prevalence often observed in environmentally sampled ticks. Finally, we were able to detect variations that help in the understanding of virus adaptations to varied environmental temperatures and mammalian hosts through a comparative approach that compares RNA folding dynamics between strains with different mammalian cell passage histories.

  • 111. Epps, Clinton W.
    et al.
    Wehausen, John D.
    Palsboll, Per J.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    McCullough, Dale R.
    Using Genetic Tools to Track Desert Bighorn Sheep Colonizations2010In: Journal of Wildlife Management, ISSN 0022-541X, E-ISSN 1937-2817, Vol. 74, no 3, p. 522-531Article in journal (Refereed)
  • 112.
    Eriksson, Jens
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Virulence Factors and Motility Mechanisms of Pathogenic Neisseria2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Neisseria gonorrhoeae and Neisseria meningitidis are two closely related human specific pathogens. Neisseria gonorrhoeae is the causative agent for the sexually transmitted disease gonorrhea and often causes asymptomatic infections in women which is a cause of infertility. Neisseria meningitidis is a major cause of mortality world-wide through bacterial meningitis and septicemia. The severity of meningococcal disease, especially in sub-Saharan Africa warrants development of effective vaccines against serogroups that currently lack them. Here, Neisseria host-pathogen interactions and common virulence factors that may prove useful in vaccine development and in understanding disease caused by pathogenic Neisseria are reviewed and investigated. The aim of this thesis is to investigate the virulence-associated properties of the universally expressed N. meningitidis proteins NhhA, NafA, PilU and PilT, as well as to characterize the twitching motility of the pathogenic Neisseria. The conserved autotransporter adhesin NhhA has in Paper I of this thesis been investigated in a murine model of meningococcemia and found to be important for intranasal colonization and disease outcome of N. meningitidis in CD46 transgenic mice. NafA has in Paper II of this thesis been named and identified as a novel anti-aggregation factor that impacts both pilus bundling and the virulence potential of N. meningitidis. The ATPases, PilU and PilT, which are involved in the functionality of pili were studied in Paper III of this thesis. PilU and PilT were found to modulate Neisseria microcolony formation, host cell adhesion, pilus retraction, serum resistance, as well as mortality in a mouse model of meningococcal disease. Finally, Paper IV of this thesis also provides novel insights into the nature of twitching motility in pathogenic Neisseria. By live-cell microscopy and automated particle tracking coupled with visualization of pili in motile bacteria we found that N. meningitidis strains, on average, move faster and utilizes more pili then N. gonorrhoeae. In summary, this thesis investigates Neisseria virulence factors in general, type IV pili in particular and characterizes the roles of several virulence-associated proteins and twitching motility in the pathogenic Neisseria.

  • 113.
    Eriksson, Jens
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Virulence Factors and Motility Mechanisms of Pathogenic Neisseria2009Licentiate thesis, monograph (Other academic)
  • 114.
    Eriksson, Jens
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Eriksson, Olaspers Sara
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Jonsson, Ann-Beth
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Loss of Meningococcal PilU Delays Microcolony Formation and Attenuates Virulence In Vivo2012In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 80, no 7, p. 2538-2547Article in journal (Refereed)
    Abstract [en]

    Neisseria meningitidis is a major cause of sepsis and bacterial meningitis worldwide. This bacterium expresses type IV pili (Tfp), which mediate important virulence traits such as the formation of bacterial aggregates, host cell adhesion, twitching motility, and DNA uptake. The meningococcal PilT protein is a hexameric ATPase that mediates pilus retraction. The PilU protein is produced from the pilT-pilU operon and shares a high degree of homology with PilT. The function of PilT in Tfp biology has been studied extensively, whereas the role of PilU remains poorly understood. Here we show that pilU mutants have delayed microcolony formation on host epithelial cells compared to the wild type, indicating that bacterium-bacterium interactions are affected. In normal human serum, the pilU mutant survived at a higher rate than that for wild-type bacteria. However, in a murine model of disease, mice infected with the pilT mutant demonstrated significantly reduced bacterial blood counts and survived at a higher rate than that for mice infected with the wild type. Infection of mice with the pilU mutant resulted in a trend of lower bacteremia, and still a significant increase in survival, than that of the wild type. In conclusion, these data suggest that PilU promotes timely microcolony formation and that both PilU and PilT are required for full bacterial virulence.

  • 115.
    Eriksson, Jens
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Eriksson, Olaspers Sara
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Palm, Oscar
    KTH.
    Sarkissian, Tim
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Wallin, Mats
    KTH.
    Jonsson, Ann-beth
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Difference in twitching motility between Neisseria meningitidis and Neisseria gonorrhoeae and its relation to pilus dynamicsManuscript (preprint) (Other academic)
    Abstract [en]

    Type IV pili of pathogenic Neisseria, i. e. Neisseria gonorrhoeae and Neisseria meningitidis, are essential for initial attachment to host cells, induction of signal transduction cascades and disease development. A characteristic feature of type IV pili is their ability to retract, which generates forces that move bacteria over surfaces. However, the relation between bacterial motility and pilus dynamics remains poorly understood. In this work we analyzed bacterial motility and monitored movement of fluorescently labeled pili by live cell imaging. We found that movement of N. meningitidis occurred at higher speed and with a larger number of retracting pili than for N. gonorrhoeae. Analysis of time-lapse images suggested that N. gonorrhoeae most often moved using one retracting pilus, whereas N. meningitidis most often used four pili. There were no differences in the membrane distribution of PilT among strains. However, we found significantly higher levels of PilT in N. gonorrhoeae than in N. meningitidis. This produces a higher retraction probability, which could contribute to explaining the lower number of pili observed in N. gonorrhoeae. Finally, we propose a mechanism for how the speed of bacterial movement on a surface depends on the number of retracting pili.

  • 116.
    Eriksson, Jesper
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Structure-Function Studies of Bacteriophage P2 Integrase and Cox protein2005Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Probably no group of organisms has been as important as bacteriophages when it comes to the understanding of fundamental biological processes like transcriptional control, DNA replication, site-specific recombination, e.t.c.

    The work presented in this thesis is a contribution towards the complete understanding of these organisms. Two proteins, integrase, and Cox, which are important for the choice of the life mode of bacteriophage P2, are investigated. P2 is a temperate phage, i.e. it can either insert its DNA into the host chromosome (by site-specific recombination) and wait (lysogeny), or it can produce new progeny with the help of the host protein machinery and thereafter lyse the cell (lytic cycle). The integrase protein is necessary for the integration and excision of the phage genome. The Cox protein is involved as a directional factor in the site-specific recombination, where it stimulates excision and inhibits integration. It has been shown that the Cox protein also is important for the choice of the lytic cycle. The choice of life mode is regulated on a transcriptional level, where two mutually exclusive promoters direct whether the lytic cycle (Pe) or lysogeny (Pc) is chosen. The Cox pro-tein has been shown to repress the Pc promoter and thereby making tran-scription from the Pe promoter possible, leading to the lytic cycle. Further, the Cox protein can function as a transcriptional activator on the parasite phage, P4. P4 has gained the ability to adopt the P2 protein machinery to its own purposes.

    In this work the importance of the native size for biologically active integrase and Cox proteins has been determined. Further, structure-function analyses of the two proteins have been performed with focus on the protein-protein interfaces. In addition it is shown that P2 Cox and the P2 relative Wphi Cox changes the DNA topology upon specific binding. From the obtained results a mechanism for P2 Cox-DNA interaction is discussed.

    The results from this thesis can be used in the development of a gene delivery system based on the P2 site-specific recombination system.

  • 117.
    Eriksson, Jesper
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Haggård-Ljungquist, Elisabeth
    The multifunctional bacteriophage Cox protein requires oligomerization for biological activity2000In: Journal of Bacteriology, ISSN 0021-9193, Vol. 182, no 23, p. 6714-6723Article in journal (Refereed)
  • 118.
    Eriksson, Jesper M
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Haggård-Ljungquist, Elisabeth
    The multifunctional bacteriophage P2 cox protein requires oligomerization for biological activity.2000In: J Bacteriol, ISSN 0021-9193, Vol. 182, no 23, p. 6714-23Article in journal (Other academic)
  • 119.
    Eriksson, Sara K
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Liu, Tao
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Haggård-Ljungquist, Elisabeth
    Interacting interfaces of the P4 antirepressor E and the P2 immunity repressor C.2000In: Mol Microbiol, ISSN 0950-382X, Vol. 36, no 5, p. 1148-55Article in journal (Other academic)
  • 120. Evers, Bastiaan
    et al.
    Helleday, Thomas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Jonkers, Jos
    Targeting homologous recombination repair defects in cancer2010In: TIPS - Trends in Pharmacological Sciences, ISSN 0165-6147, E-ISSN 1873-3735, Vol. 31, no 8, p. 372-380Article, review/survey (Refereed)
    Abstract [en]

    DNA repair is essential for cells to maintain genome stability in an environment that constantly produces DNA damage. There is a growing appreciation that defects in homologous recombination repair underlie hereditary and sporadic tumourigenesis, and that deficiency in this pathway may dictate the sensitivity of tumours to certain DNA-damaging agents. Homologous recombination deficiency (HRD) may therefore prove to be a diagnostic criterion per se if appropriate biomarkers become available to identify these tumours. In addition, homologous recombination-deficient tumours are more sensitive to inhibition of other DNA repair pathways through so-called 'synthetic lethal interactions', a principle that is currently being tested in clinical trials. Finally, homologous recombination repair-deficient cells may have an increased dependency on certain cell-cycle checkpoints, which can be therapeutically exploited. Here we describe recent advances in strategies to identify and target HRD tumours, approaches to overcome resistance, and combinatory strategies to optimize treatment outcome.

  • 121. Fattibene, Paola
    et al.
    Wojcik, Andrzej
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Biodosimetric tools for a fast triage of people accidentally exposed to ionising radiation.2009In: Annali dell'Istituto superiore di sanità, ISSN 0021-2571, Vol. 45, no 3, p. 245-Article in journal (Refereed)
  • 122.
    Fotouhi, Asal
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Skiöld, Sara
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Shakeri Manesh, Sara
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Osterman-Golkar, Siv
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Wojcik, Andrzej
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Jenssen, Dag
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Harms-Ringdahl, Mats
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Haghdoost, Siamak
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Reduction of 8-oxodGTP in the nucleotide pool by hMTH1 leads to reduction in mutations in the human lymphoblastoid cell line TK6 exposed to UVA2011In: Mutation research, ISSN 0027-5107, E-ISSN 1873-135X, Vol. 715, no 1-2, p. 13-18Article in journal (Refereed)
    Abstract [en]

    UVA has been suggested to play an important role in UV-induced mutagenesis. The mechanisms by which UVA induces mutations are still a matter of debate. Our aim was to investigate the protective capacity of hMTH1, a nucleotide pool sanitization enzyme with 8-oxodGTPase activity. Human B lymphoblastoid cells were stably transfected with shRNA directed against hMTH1. Clonogenic survival, mutations, intracellular and extracellular levels of 8-oxodG (8-oxo-7, 8-dihydro-2'-deoxyguanosine) and dG in the nucleotide pool of UVA-irradiated transfected and non-transfected cells were investigated. Mutations were determined in the thymidine kinase locus. Intracellular 8-oxodG and dG were measured using a modified ELISA and HPLC, respectively, after extraction of the nucleotide pool and conversion of nucleotides to their corresponding nucleosides. 8-oxodG in the medium was measured using ELISA. UVA-induced mutations were significantly higher while the survival was slightly lower in transfected compared to non-transfected cells. The increased mutation rate in transfected cells at increased exposure correlated with enhanced levels of 8-oxodG in the nucleotide pool, and a somewhat reduced level of 8-oxodG in the medium. The results indicate that the nucleotide pool is a significant target for UVA-induced mutations and implicates that hMTH1 plays an important role in protecting cells from UVA-induced oxidative stress.

  • 123.
    Frumerie, Clara
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Functional characterization of a phage integrase and its possible use in gene therapy2005Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Bacteriophage P2 infecting Escherichia coli can integrate into the bacterial chromosome by site-specific recombination, which is catalyzed by the P2 Int recombinase. The recombination event takes place between the phage attachment site, attP, and the bacterial attachment site, attB. Once integrated into the host chromosome the P2 prophage is very stable since an additional phage protein, Cox, is required for excision. For both integration and excision, the host-encoded protein IHF is also required.

    In this thesis, I have made a functional characterization of the P2 integrase and investigated its future potential as a tool for gene therapy. The P2 integrase was found to have cooperative interactions upon DNA binding with its accessory proteins, IHF and Cox. An N-terminal truncated Int protein retained these cooperative interactions, although it was disrupted in arm-binding. Moreover, the Int protein was found to form stable dimers in the absence of DNA, and the C-terminus and amino acid E197 was found to be important for dimerization. Dimerization was found to be essential for recombination, but dimerization deficient mutant proteins were able to bind as well as the wt protein to attP.

    The P2 Int was found to mediate recombination with a human sequence at a low frequency. It was also found that the insertion of HMG-recognition boxes can substitute for the requirement of IHF for recombination in an eukaryotic cell extract and that the integrase protein is localized in the cell nucleus. Taken together, these results indicate that the P2 integrase could be of potential use in gene therapy.

  • 124. Frumerie, Clara
    et al.
    Eriksson, Jesper
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Dugast, Marc
    Haggård-Ljungquist, Elisabeth
    Dimerization of bacteriophage P2 integrase is not required for binding to its DNA target but for its biological activity2005In: Gene, ISSN 0378-1119, Vol. 344, p. 221-231Article in journal (Refereed)
  • 125.
    Frumerie, Clara
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Eriksson, Jesper M.
    Dugast, Marc
    Haggård-Ljungquist, Elisabeth
    Dimerization of bacteriophage P2 integrase is not required for binding to its DNA target but for its biological activity2005In: Gene, ISSN 0378-1119, Vol. 344, p. 221-231Article in journal (Refereed)
  • 126.
    Frumerie, Clara
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    M Eriksson, Jesper
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Dugast, Marc
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Haggård-Ljungquist, Elisabeth
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Dimerization of bacteriophage P2 integrase is not required for binding to its DNA target but for its biological activity.2005In: Gene, ISSN 0378-1119, Vol. 344, p. 221-31Article in journal (Other academic)
  • 127.
    Frumerie, Clara
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Sylwan, Lina
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Ahlgren-Berg, Alexandra
    Haggård-Ljungquist, Elisabeth
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Cooperative interactions between bacteriophage P2 integrase and its accessory factors IHF and Cox2005In: Virology, ISSN 0042-6822, E-ISSN 1096-0341, Vol. 332, no 1, p. 284-294Article in journal (Refereed)
    Abstract [en]

    Bacteriophage P2 integrase (Int) mediates site-specific recombination leading to integration or excision of the phage genome in or out of the bacterial chromosome. Int belongs to the large family of tyrosine recombinases that have two different DNA recognition motifs binding to the arm and core sites, respectively, which are located within the phage attachment sites (attP). In addition to the P2 integrase, the accessory proteins Escherichia coli IHF and P2 Cox are needed for recombination. IHF is a structural protein needed for integration and excision by bending the DNA. As opposed to lambda, only one IHF site is found in P2 attP. P2 Cox controls the direction of recombination by inhibiting integration but being required for excision. In this work, the effects of accessory proteins on the capacity of Int to bind to its DNA recognition sequences are analyzed using electromobility shifts. P2 Int binds with low affinity to the arm site, and this binding is greatly enhanced by IHF. The arm binding domain of Int is located at the N-terminus. P2 Int binds with high affinity to the core site, and this binding is also enhanced by IHF. The fact that the cooperative binding of Int and IHF is strongly reduced by lengthening the distance between the IHF and core binding sites indicates that the distance between these sites may be important for cooperative binding. The Int and Cox proteins also bind cooperatively to attP.

  • 128.
    Frumerie, Clara
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Sylwan, Lina
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Helleday, Thomas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Yu, Anna
    Department of Clinical Chemistry, Danderyd Hospital and Karolinska Institute, Stockholm, Sweden.
    Haggård-Ljungquist, Elisabeth
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Bacteriophage P2 integrase: another possible tool for site-specific recombination in eukaryotic cells2008In: Journal of Applied Microbiology, ISSN 1364-5072, E-ISSN 1365-2672, Vol. 105, no 1, p. 290-299Article in journal (Refereed)
    Abstract [en]

    AIMS: To investigate if the site-specific tyrosine integrase (Int) from phage P2 has features that would make it interesting for use of gene transfer into eukaryotic cells. These include the possibility of promoting recombination with a nonphage sequence, abolishing the requirement for the bacterial DNA-binding and -bending protein integration host factor (IHF), and localization to the nucleus of eukaryotic cells. METHODS AND RESULTS: We show that the Int protein catalyzes site-specific recombination using a human sequence in Escherichia coli and in vitro although not as efficiently as with the wild-type bacterial sequence, and that insertion of high mobility group recognition boxes in the phage attachment site substrate abolish the requirement of IHF and allows efficient recombination in vitro in a eukaryotic cell extract. Furthermore, we show by fluorescence that the Int protein contains a functional intrinsic nuclear localization signal, localizing it to the nucleus in both HeLa and 293 cells. CONCLUSIONS: We conclude that P2 Int may be a potential tool for site-specific integration of genes into the human chromosome. SIGNIFICANCE AND IMPACT OF THE STUDY: The study implies the possibility of using multiple prokaryotic Int proteins with different specific integration sites in human cells for future gene therapy programmes.

  • 129. Gad, Helge
    et al.
    Svensson, Linda M.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Saleh, Aljona
    Stockholm University, Faculty of Science, Department of Analytical Chemistry.
    Berntsson, Ronnie P.-A.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gustafsson, Robert
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Johansson, Fredrik
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Djureinovic, Tatjana
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Häggblad, Maria
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Martens, Ulf
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Lundgren, Bo
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Granelli, Ingrid
    Stockholm University, Faculty of Science, Department of Analytical Chemistry.
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Helleday, Thomas
    MTH1 inhibition eradicates cancer by preventing sanitation of the dNTP pool2014In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 508, no 7495, p. 215-221Article in journal (Refereed)
    Abstract [en]

    Cancers have dysfunctional redox regulation resulting in reactive oxygen species production, damaging both DNA and free dNTPs. The MTH1 protein sanitizes oxidized dNTP pools to prevent incorporation of damaged bases during DNA replication. Although MTH1 is non-essential in normal cells, we show that cancer cells require MTH1 activity to avoid incorporation of oxidized dNTPs, resulting in DNA damage and cell death. We validate MTH1 as an anticancer target in vivo and describe small molecules TH287 and TH588 as first-in-class nudix hydrolase family inhibitors that potently and selectively engage and inhibit the MTH1 protein in cells. Protein co-crystal structures demonstrate that the inhibitors bind in the active site of MTH1. The inhibitors cause incorporation of oxidized dNTPs in cancer cells, leading to DNA damage, cytotoxicity and therapeutic responses in patient-derived mouse xenografts. This study exemplifies the non-oncogene addiction concept for anticancer treatment and validates MTH1 as being cancer phenotypic lethal.

  • 130. Gelius, Eva
    et al.
    Persson, Carina
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Karlsson, Jenny
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Steiner, Håkan
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    A mammalian peptidoglycan recognition protein with N-acetylmuramoyl-L-alanine amidase activity2003In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 306, no 4, p. 988-994Article in journal (Refereed)
    Abstract [en]

    The family of peptidoglycan recognition proteins (PGRPs) is conserved from insects to mammals. Recently, Drosophila PGRP-SC1B was demonstrated to be an N-acetylmuramoyl- -alanine amidase (NAMLAA), an enzyme that cleaves the lactylamide bond between muramic acid and the peptide chain in peptidoglycan (PGN). We now show an M.mPGRP-L mRNA to be expressed in the liver. The recombinant M.mPGRP-L protein has NAMLAA activity and degrades PGN from both Escherichia coli and Staphylococcus aureus; however, the Gram-positive PGN was a better substrate after lysozyme treatment. The activity of M.mPGRP-L was further analysed using Bordetella pertussis tracheal toxin as a substrate. Cleavage products were separated on HPLC and identified using mass spectrometry. From these results we conclude that M.mPGRP-L has activity and other properties identifying it as the NAMLAA protein present in mammalian sera.

  • 131.
    Geörg, Miriam
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Interactions between pathogenic bacteria and host cells2011Licentiate thesis, monograph (Other academic)
  • 132.
    Gonzalez de Valdivia, Ernesto I.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Studies on translation initiation and gene expression in Escherichia coli2006Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    In prokaryotes, several mRNA sequences surrounding the initiation codon have been found to influence the translation process; these include the downstream region and its codon context, the Shine-Dalgarno sequence and the S1 ribosomal protein-binding site. In this thesis, the purpose has been to study the role of the downstream region and Shine-Dalgarno-like sequences on early translation elongation and gene expression in Escherichia coli.

    The downstream region (DR) after the initiation codon (around five to seven codons), has an important role in the initiation of translation. We find that most of the codons which give very low gene expression at +2 (considering AUG as +1), reach 5 to 10 fold higher expression when those codons are positioned posteriori to +2, with the exception of the NGG codons. The NGG codons abort the translation process if located within the first five codons of the DR, due to peptidyl-tRNA drop-off. However, when the NGG codons are situated further down from the DR, the protein expression was increased at the same level of expression as in the presence of any other codon.

    The Shine-Dalgarno (SD) is an important region of initiation in translation of bacteria. In spite of this, it has been found that Gram-negative bacteria could translate mRNAs with weak or non-functional SD, while the DR carries out a main role in the efficiency of translation. In addition, positions of SD and SD-like sequences are very important to direct initiation of translation in the choice between two possible initiation codons. A strong SD between two initiation sites will favor the second initiation site if it consists of a canonical start codon followed by a good DR.

    The results suggest that the mRNA sequences surrounding the initiation codon: the downstream region and the Shine-Dalgarno and SD-like sequences, are very important contributors to the translation level and gene expression in Escherichia coli.

  • 133.
    Gonzalez de Valdivia, Ernesto I.
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Isaksson, Leif A.
    A codon window in mRNA downstream of the initiation codon where NGG codons give strongly reduced gene expression in Escherichia coli2004In: Nucleic Acids Reseach, Vol. 32, no 17, p. 5198-5205Article in journal (Refereed)
  • 134.
    Gonzalez de Valdivia, Ernesto I.
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Isaksson, Leif A.
    Abortive translation caused by peptidyl-tRNA drop-off at NGG codons in the early coding region of mRNA2005In: FEBS J, Vol. 272, no 20, p. 5306-5316Article in journal (Refereed)
  • 135. Gottipati, Ponnari
    et al.
    Cassel, Tobias N.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Savolainen, Linda
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Helleday, Thomas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Transcription-associated recombination is dependent on replication in Mammalian cells2008In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 28, no 1, p. 154-64Article in journal (Refereed)
    Abstract [en]

    Transcription can enhance recombination; this is a ubiquitous phenomenon from prokaryotes to higher eukaryotes. However, the mechanism of transcription-associated recombination in mammalian cells is poorly understood. Here we have developed a construct with a recombination substrate in which levels of recombination can be studied in the presence or absence of transcription. We observed a direct enhancement in recombination when transcription levels through the substrate were increased. This increase in homologous recombination following transcription is locus specific, since homologous recombination at the unrelated hprt gene is unaffected. In addition, we have shown that transcription-associated recombination involves both short-tract and long-tract gene conversions in mammalian cells, which are different from double-strand-break-induced recombination events caused by endonucleases. Transcription fails to enhance recombination in cells that are not in the S phase of the cell cycle. Furthermore, inhibition of transcription suppresses induction of recombination at stalled replication forks, suggesting that recombination may be involved in bypassing transcription during replication.

  • 136. Gottipati, Ponnari
    et al.
    Helleday, Thomas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Transcription-associated recombination in eukaryotes: link between transcription, replication and recombination.2009In: Mutagenesis, ISSN 1464-3804, Vol. 24, no 3, p. 203-10Article in journal (Refereed)
    Abstract [en]

    Homologous recombination (HR) is an important DNA repair pathway and is essential for cellular survival. It plays a major role in repairing replication-associated lesions and is functionally connected to replication. Transcription is another cellular process, which has emerged to have a connection with HR. Transcription enhances HR, which is a ubiquitous phenomenon referred to as transcription-associated recombination (TAR). Recent evidence suggests that TAR plays a role in inducing genetic instability, for example in the THO mutants (Tho2, Hpr1, Mft1 and Thp2) in yeast or during the development of the immune system leading to genetic diversity in mammals. On the other hand, evidence also suggests that TAR may play a role in preventing genetic instability in many different ways, one of which is by rescuing replication during transcription. Hence, TAR is a double-edged sword and plays a role in both preventing and inducing genetic instability. In spite of the interesting nature of TAR, the mechanism behind TAR has remained elusive. Recent advances in the area, however, suggest a link between TAR and replication and show specific genetic requirements for TAR that differ from regular HR. In this review, we aim to present the available evidence for TAR in both lower and higher eukaryotes and discuss its possible mechanisms, with emphasis on its connection with replication.

  • 137. Gottipati, Ponnari
    et al.
    Vischioni, Barbara
    Schultz, Niklas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Solomons, Joyce
    Bryant, Helen E.
    Djureinovic, Tatjana
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Issaeva, Natalia
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Sleeth, Kate
    Sharma, Ricky A.
    Helleday, Thomas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Poly(ADP-Ribose) Polymerase Is Hyperactivated in Homologous Recombination-Defective Cells2010In: Cancer Research, ISSN 0008-5472, E-ISSN 1538-7445, Vol. 70, no 13, p. 5389-5398Article in journal (Refereed)
    Abstract [en]

    Poly(ADP-ribose) (PAR) polymerase 1 (PARP1) is activated by DNA single-strand breaks (SSB) or at stalled replication forks to facilitate DNA repair. Inhibitors of PARP efficiently kill breast, ovarian, or prostate tumors in patients carrying hereditary mutations in the homologous recombination (HR) genes BRCA1 or BRCA2 through synthetic lethality. Here, we surprisingly show that PARP1 is hyperactivated in replicating BRCA2-defective cells. PARP1 hyperactivation is explained by the defect in HR as shRNA depletion of RAD54, RAD52, BLM, WRN, and XRCC3 proteins, which we here show are all essential for efficient HR and also caused PARP hyperactivation and correlated with an increased sensitivity to PARP inhibitors. BRCA2-defective cells were not found to have increased levels of SSBs, and PAR polymers formed in HR-defective cells do not colocalize to replication protein A or gamma H2AX, excluding the possibility that PARP hyperactivity is due to increased SSB repair or PARP induced at damaged replication forks. Resistance to PARP inhibitors can occur through genetic reversion in the BRCA2 gene. Here, we report that PARP inhibitor-resistant BRCA2-mutant cells revert back to normal levels of PARP activity. We speculate that the reason for the sensitivity of HR-defective cells to PARP inhibitors is related to the hyperactivated PARP1 in these cells. Furthermore, the presence of PAR polymers can be used to identify HR-defective cells that are sensitive to PARP inhibitors, which may be potential biomarkers. Cancer Res; 70(13); 5389-98. (C) 2010 AACR.

  • 138. Gradecka-Meesters, Dobroslawa
    et al.
    Palus, Jadwiga
    Prochazka, Gabriela
    Segerback, Dan
    Dziubaltowska, Elzbieta
    Kotova, Natalia
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Jenssen, Dag
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Arkusz, Joanna
    Lundin, Cecilia
    Vikstrom, Elisabet
    Rydzynski, Konrad
    Nilsson, Robert
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Stepnik, Maciej
    Assessment of the protective effects of selected dietary anticarcinogens against DNA damage and cytogenetic effects induced by benzo[a]pyrene in C57BL/6J mice2011In: Food and Chemical Toxicology, ISSN 0278-6915, E-ISSN 1873-6351, Vol. 49, no 8, p. 1674-1683Article in journal (Refereed)
    Abstract [en]

    The protective action in C57BL/6J mice from orally administered ellagic acid (EA), benzyl isothiocyanate (BITC), an extract of epigallocatechins (Tegreen (R)) as well as chlorophyllin (CHL) against benzo[a]pyrene (B[a]P)-induced DNA damage and cytogenetic effects was investigated. In pilot experiment the comet assay indicated protective effects for all compounds, while such activity was confined to EA and CH with respect to B[a]P-DNA adducts and micronuclei. EA and CH were chosen for the main study where the levels of DNA adducts in liver after injection of 30 mg B[a]P/kg b.w. did not differ from those found for animals exposed to B[a]P and treated with the protective substances. In leukocytes no significant protective effect of CHL was detected while a 2-fold increase of adduct concentrations was observed after co-administration of EA. In the comet assay CHL or EA caused a 3-fold decrease of SSB, and a 2-fold decrease of FPG sites in comparison to animals treated with B[a]P. CHL or EA showed a significant protective effect against B[a]P-induced MN in polychromatic erythrocytes in bone marrow. In contrast, flow cytometry measurements in peripheral blood indicated the MN frequency after treatment with CHL or EA almost twice as high as that recorded for B[a]P alone.

  • 139.
    Groth, Petra
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Efficient restart of MMS stalled replication forks is independent of recombination or base excision repair and leave post replication damageManuscript (preprint) (Other academic)
  • 140.
    Groth, Petra
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Maintaining DNA replication fork integrity2009Licentiate thesis, monograph (Other academic)
  • 141.
    Groth, Petra
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Replication Dynamics in the DNA Damage Response2011Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Faithful DNA replication is essential and the induction of replication stress may have profound effects on genomic integrity. This is demonstrated by the formation of DNA double strand breaks (DSBs), considered to be the most toxic DNA lesions, at stalled replication forks. Homologous recombination (HR) has been shown to be involved in the replication stress response and has been suggested for stabilisation, restart and repair of stalled replication forks. However, the HR mechanisms induced by replication stress are still, to a major part, unknown. The present thesis focuses on investigating replication patterns following the induction of replication stress. Further, the consequences of stressed replication are studied by detection of DSB formation and characterisation of HR in mammalian cells.

    Here, we have identified WEE1, a regulator of mitotic entry, as a factor required to maintain correct replication. Depletion of WEE1 results in the formation of DSBs specifically in newly replicated DNA, as visualised in a modified pulse field electrophoresis assay. We were also able to detect formation of replication-associated secondary DSBs following treatment with ionizing radiation (IR). These DSBs were further demonstrated as major substrates for IR induced HR.

    Using the DNA fibre technique we investigated the effect of DNA alkylating agents on replication. We found that DNA methylations pose direct physical blocks to progressing replication forks causing them to stall in a checkpoint independent manner. Furthermore, we studied restart kinetics following methylation blocked replication and identified a distinct restart mechanism for blocked replication forks independent of new origin firing and HR.

    In conclusion, our findings increase the knowledge of replication dynamics following perturbed replication and further clarify the role of HR following IR induced damage and DNA alkylation.

  • 142.
    Groth, Petra
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Auslander, Simon
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Majumder, Muntasir Mamun
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Schultz, Niklas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Johansson, Fredrik
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Petermann, Eva
    Helleday, Thomas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Methylated DNA Causes a Physical Block to Replication Forks Independently of Damage Signalling, O-6-Methylguanine or DNA Single-Strand Breaks and Results in DNA Damage2010In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 402, no 1, p. 70-82Article in journal (Refereed)
    Abstract [en]

    Even though DNA alkylating agents have been used for many decades in the treatment of cancer, it remains unclear what happens when replication forks encounter alkylated DNA. Here, we used the DNA fibre assay to study the impact of alkylating agents on replication fork progression. We found that the alkylator methyl methanesulfonate (MMS) inhibits replication elongation in a manner that is dose dependent and related to the overall alkylation grade. Replication forks seem to be completely blocked as no nucleotide incorporation can be detected following 1 h of MMS treatment. A high dose of 5 mM caffeine, inhibiting most DNA damage signalling, decreases replication rates overall but does not reverse MMS-induced replication inhibition, showing that the replication block is independent of DNA damage signalling. Furthermore, the block of replication fork progression does not correlate with the level of DNA single-strand breaks. Overexpression of O-6-methylguanine (O6meG)-DNA methyltransferase protein, responsible for removing the most toxic alkylation, O6meG, did not affect replication elongation following exposure to N-methyl-M-nitro-N-nitrosoguanidine. This demonstrates that O6meG lesions are efficiently bypassed in mammalian cells. In addition, we find that MMS-induced gamma H2AX foci co-localise with 53BP1 foci and newly replicated areas, suggesting that DNA double-strand breaks are formed at MMS-blocked replication forks. Altogether, our data suggest that N-alkylations formed during exposure to alkylating agents physically block replication fork elongation in mammalian cells, causing formation of replication-associated DNA lesions, likely double-strand breaks.

  • 143.
    Groth, Petra
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Elvers, Ingegerd
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Orta, M. L.
    Majumder, M. M.
    Lagerqvist, Anne
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Helleday, Thomas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Ionizing radiation induces replication-associated secondary DNA double-strand breaks that are substrates for homologous recombination repairManuscript (preprint) (Other academic)
  • 144.
    Groth, Petra
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Luis Orta, Manuel
    Elvers, Ingegerd
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Majumder, Muntasir Mamun
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Lagerqvist, Anne
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Helleday, Thomas
    Homologous recombination repairs secondary replication induced dna double strand breaks after ionizing radiation2012In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 40, no 14, p. 6585-6594Article in journal (Refereed)
    Abstract [en]

    Ionizing radiation (IR) produces direct two-ended DNA double-strand breaks (DSBs) primarily repaired by non-homologous end joining (NHEJ). It is, however, well established that homologous recombination (HR) is induced and required for repair of a subset of DSBs formed following IR. Here, we find that HR induced by IR is drastically reduced when post-DNA damage replication is inhibited in mammalian cells. Both IR-induced RAD51 foci and HR events in the hprt gene are reduced in the presence of replication polymerase inhibitor aphidicolin (APH). Interestingly, we also detect reduced IR-induced toxicity in HR deficient cells when inhibiting post-DNA damage replication. When studying DSB formation following IR exposure, we find that apart from the direct DSBs the treatment also triggers formation of secondary DSBs peaking at 7-9 h after exposure. These secondary DSBs are restricted to newly replicated DNA and abolished by inhibiting post-DNA damage replication. Further, we find that IR-induced RAD51 foci are decreased by APH only in cells replicating at the time of IR exposure, suggesting distinct differences between IR-induced HR in S- and G2-phases of the cell cycle. Altogether, our data indicate that secondary replication-associated DSBs formed following exposure to IR are major substrates for IR-induced HR repair.

  • 145.
    Gubanova, Evgenia
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    The role of SMG-1 in the DNA damage response2011Licentiate thesis, monograph (Other academic)
  • 146.
    Gubanova, Evgenia
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Brown, Brandee
    Ivanov, Sergei V.
    Helleday, Thomas
    Mills, Gordon B.
    Yarbrough, Wendell G.
    Issaeva, Natalia
    Downregulation of SMG-1 in HPV-Positive Head and Neck Squamous Cell Carcinoma Due to Promoter Hypermethylation Correlates with Improved Survival2012In: Clinical Cancer Research, ISSN 1078-0432, E-ISSN 1557-3265, Vol. 18, no 5, p. 1257-1267Article in journal (Refereed)
    Abstract [en]

    Purpose: Human papillomavirus (HPV) is linked with a subset of head and neck squamous cell carcinomas (HNSCC). HPV-positive HNSCCs show a better prognosis than HPV-negative HNSCCs, which may be explained by sensitivity of the HPV-positive HNSCCs to ionizing radiation (IR). Although the molecular mechanism behind sensitivity to IR in HPV-positive HNSCCs is unresolved, DNA damage response (DDR) might be a significant determinant of IR sensitivity. An important player in the DDR, SMG-1 (suppressor with morphogenetic effect on genitalia), is a potential tumor suppressor and may therefore be deregulated in cancer. No studies have yet been conducted linking defects in SMG-1 expression with cancer. We investigated whether deregulation of SMG-1 could be responsible for defects in the DDR in oropharyngeal HNSCC. Experimental Design: Expression and promoter methylation status of SMG-1 were investigated in HNSCCs. To identify a functional link between HPV infection and SMG-1, we transfected the HPV-negative cells with an E6/E7 expression construct. SMG-1 short hairpin RNAs were expressed in HPV-negative cells to estimate survival upon IR. Results: Forced E6/E7 expression in HPV-negative cells resulted in SMG-1 promoter hypermethylation and decreased SMG-1 expression. Due to promoter hypermethylation, HPV-positive HNSCC cells and tumors express SMG-1 at lower levels than HPV-negative SCCs. Depletion of SMG-1 in HPV-negative HNSCC cells resulted in increased radiation sensitivity, whereas SMG-1 overexpression protected HPV-positive tumor cells from irradiation. Conclusions: Levels of SMG-1 expression negatively correlated with HPV status in cancer cell lines and tumors. Diminished SMG-1 expression may contribute to the enhanced response to therapy exhibited by HPV-positive HNSCCs. 

  • 147.
    Gubanova, Evgenia
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. Science for Life Laboratory; Division of Translational Medicine and Chemical Biology; Department of Medical Biochemistry and Biophysics; Karolinska Institut; Stockholm, Sweden.
    Issaeva, Natalia
    Djureinovic, Tatjana
    Helleday, Thomas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    SMG-1 regulates senescence and suppresses epithelial-mesenchymal transitionManuscript (preprint) (Other academic)
  • 148. Haeuser, Roman
    et al.
    Blasche, Sonja
    Dokland, Terje
    Haggård-Ljungquist, Elisabeth
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    von Brunn, Albrecht
    Salas, Margarita
    Casjens, Sherwood
    Molineux, Ian
    Uetz, Peter
    Bacteriophage Protein-Protein Interactions2012In: Advances in virus research, vol 83: Bacteriophages, pt B, ELSEVIER ACADEMIC PRESS , 2012, p. 219-298Chapter in book (Refereed)
    Abstract [en]

    Bacteriophages T7, lambda, P22, and P2/P4 (from Escherichia coli), as well as phi 29 (from Bacillus subtilis), are among the best-studied bacterial viruses. This chapter summarizes published protein interaction data of intraviral protein interactions, as well as known phage-host protein interactions of these phages retrieved from the literature. We also review the published results of comprehensive protein interaction analyses of Pneumococcus phages Dp-1 and Cp-1, as well as coliphages lambda and T7. For example, the approximate to 55 proteins encoded by the T7 genome are connected by approximate to 43 interactions with another approximate to 15 between the phage and its host. The chapter compiles published interactions for the well-studied phages lambda (33 intra-phage/22 phage-host), P22 (38/9), P2/P4 (14/3), and phi 29 (20/2). We discuss whether different interaction patterns reflect different phage lifestyles or whether they may be artifacts of sampling. Phages that infect the same host can interact with different host target proteins, as exemplified by E. coli phage lambda and T7. Despite decades of intensive investigation, only a fraction of these phage interactomes are known. Technical limitations and a lack of depth in many studies explain the gaps in our knowledge. Strategies to complete current interactome maps are described. Although limited space precludes detailed overviews of phage molecular biology, this compilation will allow future studies to put interaction data into the context of phage biology.

  • 149.
    Haggård-Ljungquist, Elisabeth
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Jacobsen, Erik
    Rishovd, Svein
    Six, Erich W
    Nilssen, Öivind
    Sunshine, Melvin G
    Lindqvist, Björn H
    Kim, Kyoung-Jin
    Barreiro, Virginia
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Koonin, Eugene V
    Bacteriophage P2: genes involved in baseplate assembly.1995In: Virology, ISSN 0042-6822, Vol. 213, no 1, p. 109-21Article in journal (Other academic)
  • 150.
    Haghdoost, Siamak
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Biomarkers of oxidative stress and their application for assessment of individual radiosensitivity2005Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Radiotherapy is one of the most common therapeutic methods for treatment of many types of cancer. Despite many decades of development and experience there is much to improve, both in efficacy of treatment and to decrease the incidences of adverse healthy tissue reactions. Around 20 % of the radiotherapy patients show a broad range in the severity of normal tissue reactions to radiotherapy, and dose limits are governed by severe reactions in the most radiosensitive patients (< 5 %). Identification of patients with low, moderate or high clinical radiosensitivity before commencing of radiotherapy would allow individual adaptation of the maximum dose with an overall increase in the cure rate. Characterization of factors that may modify the biological effects of ionizing radiation has been a subject of intense research efforts. Still, there is no assay currently available that can reliably predict the clinical radiosensitivity. The aim of this work has been to investigate the role of oxidative stress in individual radiosensitivity and evaluate novel markers of radiation response, which could be adapted for clinical use.

    8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG), a general marker of oxidative stress, is one of the major products of interaction of ionizing radiation with DNA and the nucleotide pool of the cell. As 8-oxo-dG is highly mutagenic due to incorrect base pairing with deoxyadenosine, various repair mechanisms recognize and remove 8-oxo-dG. The repaired lesions are released from cells to the extracellular milieu (serum, urine and cell culture medium) where they can be detected as markers for free radical reactions with the nucleic acids.

    Significant variations in background levels as well as in radiation induced levels of 8-oxo-dG in urine have been demonstrated in breast cancer patients (paper 1). Two major patterns were observed: high background and no therapy-related increase vs. low background and significant increase during radiotherapy for the radiosensitive and non radiosensitive patients respectively.

    Studies in paper 2 indicated major contribution of the nucleotide pool to the extracellular 8-oxo-dG levels. The results also implicated induction of prolonged endogenous oxidative stress in the irradiated cells. RNA “knock-down” experiments on the nucleotide pool sanitization enzyme hMTH1 in paper 3 lend further experimental evidence to this assumption.

    The applicability of 8-oxo-dG as a diagnostic marker of oxidative stress was demonstrated in paper 4. Studies on dialysis patients revealed a good correlation between inflammatory responses (known to be associated with persistent oxidative stress) and extracellular 8-oxo-dG.

    In summary, our results confirm that extracellular 8-oxo-dG is a sensitive in vivo biomarker of oxidative stress, primarily formed by oxidative damage of dGTP in the nucleotide pool with a potential to become a clinical tool for prediction of individual responses to radiotherapy.

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