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  • 1. Al-Minawi, Ali Z.
    et al.
    Lee, Yin-Fai
    Håkansson, Daniel
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Johansson, Fredrik
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Lundin, Cecilia
    Saleh-Gohari, Nasrollah
    Schultz, Niklas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Jenssen, Dag
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Bryant, Helen E.
    Meuth, Mark
    Hinz, John M.
    Helleday, Thomas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    The ERCC1/XPF endonuclease is required for completion of homologous recombination at DNA replication forks stalled by inter-strand cross-links2009In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 37, no 19, p. 6400-6413Article in journal (Refereed)
    Abstract [en]

    Both the ERCC1-XPF complex and the proteins involved in homoIogous recombination (HR) have critical roles in inter-strand cross-link (ICL) repair. Here, we report that mitomycin C-induced lesions inhibit replication fork elongation. Furthermore, mitomycin C-induced DNA double-strand breaks (DSBs) are the result of the collapse of ICL-stalled replication forks. These are not formed through replication run off, as we show that mitomycin C or cisplatin-induced DNA lesions are not incised by global genome nucleotide excision repair (GGR). We also suggest that ICL-lesion repair is initiated either by replication or transcription, as the GGR does not incise ICL-lesions. Furthermore, we report that RAD51 foci are induced by cisplatin or mitomycin C independently of ERCC1, but that mitomycin C-induced HR measured in a reporter construct is impaired in ERCC1-defective cells. These data suggest that ERCC1-XPF plays a role in completion of HR in ICL repair. We also find no additional sensitivity to cisplatin by siRNA co-depletion of XRCC3 and ERCC1, showing that the two proteins act on the same pathway to promote survival.

  • 2. Bartkova, Jirina
    et al.
    Rezaei, Nousin
    Liontos, Michalis
    Karakaidos, Panagiotis
    Kletsas, Dimitris
    Issaeva, Natalia
    Stockholm University.
    Vassiliou, Leandros-Vassilios F
    Kolettas, Evangelos
    Niforou, Katerina
    Zoumpourlis, Vassilis C
    Takaoka, Munenori
    Nakagawa, Hiroshi
    Tort, Frederic
    Fugger, Kasper
    Johansson, Fredrik
    Stockholm University.
    Sehested, Maxwell
    Andersen, Claus L
    Dyrskjot, Lars
    Ørntoft, Torben
    Lukas, Jiri
    Kittas, Christos
    Helleday, Thomas
    Stockholm University.
    Halazonetis, Thanos D
    Bartek, Jiri
    Gorgoulis, Vassilis G
    Oncogene-induced senescence is part of the tumorigenesis barrier imposed by DNA damage checkpoints.2006In: Nature, ISSN 1476-4687, Vol. 444, no 7119, p. 633-7Article in journal (Refereed)
  • 3.
    Biverstal, Anna
    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.
    Jenssen, Dag
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Erixon, Klaus
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Cyclobutane pyrimidine dimers do not fully explain the mutagenicity induced by UVA in Chinese hamster cells2008In: Mutation research, ISSN 0027-5107, E-ISSN 1873-135X, Vol. 648, no 02-jan, p. 32-39Article in journal (Refereed)
    Abstract [en]

    UVA generates low levels of cyclobutane pyrimidine dimers (CPDs). Here we asked the question whether CPDs could fully explain the level of mutations induced by UVA. Relative mutagenicities of UVA and UVC were calculated at equal levels of CPDs in cell lines, deficient in different aspects of repair. Survival and gene mutations in the hprt locus were analyzed in a set of Chinese hamster ovary (CHO) cell lines, i.e., wild-type, Cockayne syndrome B protein-deficient (CSB), XRCC3-deficient and XRCC1-deficient adjusted to the same level of CPDs which was analyzed as strand breaks as a result of DNA cleavage by T4 endonuclease V at CPD sites. Induced mutagenicity of UVA was approximately 2 times higher than the mutagenicity of UVC in both wild-type and XRCC1-deficient cells when calculated at equal level of CPDs. Since this discrepancy could be explained by the fact that the TT-dimers, induced by UVA, might be more mutagenic than C-containing CPDs induced by UVC, we applied acetophenone, a photosensitizer previously shown to generate enhanced levels of TT-CPDs upon UVB exposure. The results suggested that the TT-CPDs were actually less mutagenic than the C-containing CPDs. We also found that the mutagenic effect of UVA was not significantly enhanced in a cell line deficient in the repair of CPDs. Altogether this suggests that neither base excision- nor nucleotide excision-repair was involved. We further challenge the possibility that the lesion responsible for the mutations induced by UVA was of a more complex nature and which possibly is repaired by homologous recombination (HR). The results indicated that UVA was more recombinogenic than UVC at equal levels of CPDs. We therefore suggest that UVA induces a complex type of lesion, which might be an obstruction during replication fork progression that requires HR repair to be further processed. 

  • 4. Bryant, Helen E
    et al.
    Petermann, Eva
    Schultz, Niklas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Jemth, Ann-Sofie
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Loseva, Olga
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Issaeva, Natalia
    Johansson, Fredrik
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Fernandez, Serena
    McGlynn, Peter
    Helleday, Thomas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    PARP is activated at stalled forks to mediate Mre11-dependent replication restart and recombination.2009In: The EMBO journal, ISSN 1460-2075, Vol. 28, no 17, p. 2601-15Article in journal (Refereed)
    Abstract [en]

    If replication forks are perturbed, a multifaceted response including several DNA repair and cell cycle checkpoint pathways is activated to ensure faithful DNA replication. Here, we show that poly(ADP-ribose) polymerase 1 (PARP1) binds to and is activated by stalled replication forks that contain small gaps. PARP1 collaborates with Mre11 to promote replication fork restart after release from replication blocks, most likely by recruiting Mre11 to the replication fork to promote resection of DNA. Both PARP1 and PARP2 are required for hydroxyurea-induced homologous recombination to promote cell survival after replication blocks. Together, our data suggest that PARP1 and PARP2 detect disrupted replication forks and attract Mre11 for end processing that is required for subsequent recombination repair and restart of replication forks.

  • 5. El-Khamisy, Sherif F
    et al.
    Saifi, Gulam M
    Weinfeld, Michael
    Johansson, Fredrik
    Stockholm University.
    Helleday, Thomas
    Stockholm University.
    Lupski, James R
    Caldecott, Keith W
    Defective DNA single-strand break repair in spinocerebellar ataxia with axonal neuropathy-1.2005In: Nature, ISSN 1476-4687, Vol. 434, no 7029, p. 108-13Article in journal (Refereed)
  • 6.
    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.

  • 7.
    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. 

  • 8.
    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)
  • 9.
    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.

  • 10.
    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.

  • 11.
    Helleday, T
    et al.
    Stockholm University.
    Johansson, F
    Stockholm University.
    Jenssen, D
    Stockholm University.
    The DRAG test: an assay for detection of genotoxic damage.2001In: Altern Lab Anim, ISSN 0261-1929, Vol. 29, no 3, p. 233-41Article in journal (Refereed)
  • 12. Jansen, Jacob G.
    et al.
    Tsaalbi-Shtylik, Anastasia
    Hendriks, Giel
    Gali, Himabindu
    Hendel, Ayal
    Johansson, Fredrik
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Erixon, Klaus
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Livneh, Zvi
    Mullenders, Leon H. F.
    Haracska, Lajos
    de Wind, Niels
    Separate Domains of Rev1 Mediate Two Modes of DNA Damage Bypass in Mammalian Cells2009In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 29, no 11, p. 3113-3123Article in journal (Refereed)
    Abstract [en]

    The Y family DNA polymerase Rev1 has been proposed to play a regulatory role in the replication of damaged templates. To elucidate the mechanism by which Rev1 promotes DNA damage bypass, we have analyzed the progression of replication on UV light-damaged DNA in mouse embryonic fibroblasts that contain a defined deletion in the N-terminal BRCT domain of Rev1 or that are deficient for Rev1. We provide evidence that Rev1 plays a coordinating role in two modes of DNA damage bypass, i.e., an early and a late pathway. The cells carrying the deletion in the BRCT domain are deficient for the early pathway, reflecting a role of the BRCT domain of Rev1 in mutagenic translesion synthesis. Rev1-deficient cells display a defect in both modes of DNA damage bypass. Despite the persistent defect in the late replicational bypass of fork-blocking (6-4) pyrimidine-pyrimidone photoproducts, overall replication is not strongly affected by Rev1 deficiency. This results in almost completely replicated templates that contain gaps encompassing the photoproducts. These gaps are inducers of DNA damage signaling leading to an irreversible G(2) arrest. Our results corroborate a model in which Rev1-mediated DNA damage bypass at postreplicative gaps quenches irreversible DNA damage responses.

  • 13.
    Johansson, Fredrik
    et al.
    Stockholm University.
    Allkvist, Annika
    Erixon, Klaus
    Stockholm University.
    Malmvärn, Anna
    Stockholm University.
    Nilsson, Robert
    Stockholm University.
    Bergman, Ake
    Helleday, Thomas
    Stockholm University.
    Jenssen, Dag
    Stockholm University.
    Screening for genotoxicity using the DRAG assay: investigation of halogenated environmental contaminants.2004In: Mutat Res, ISSN 0027-5107, Vol. 563, no 1, p. 35-47Article in journal (Refereed)
  • 14.
    Johansson, Fredrik
    et al.
    Stockholm University.
    Lagerqvist, Anne
    Stockholm University.
    Filippi, Silvia
    Palitti, Fabrizio
    Erixon, Klaus
    Stockholm University.
    Helleday, Thomas
    Stockholm University.
    Jenssen, Dag
    Stockholm University.
    Caffeine delays replication fork progression and enhances UV-induced homologous recombination in Chinese hamster cell lines.2006In: DNA Repair (Amst), ISSN 1568-7864, Vol. 5, no 12, p. 1449-58Article in journal (Refereed)
  • 15. Matsuoka, Atsuko
    et al.
    Lundin, Cecilia
    Johansson, Fredrik
    Sahlin, Margareta
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Fukuhara, Kiyoshi
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Jenssen, Dag
    Önfelt, Agneta
    Correlation of sister chromatid exchange formation through homologous recombination with ribonucleotide reductase inhibition.2004In: Mutat Res, ISSN 0027-5107, Vol. 547, no 1-2, p. 101-7Article in journal (Other academic)
    Abstract [en]

    We conducted the recombination and sister chromatid exchange (SCE) assays with five chemicals (hydroxyurea (HU), resveratrol, 4-hydroxy-trans-stilbene, 3-hydroxy-trans-stilbene, and mitomycin C) in Chinese hamster cell line SPD8/V79 to confirm directly that SCE is a result of homologous recombination (HR). SPD8 has a partial duplication in exon 7 of the endogenous hprt gene and can revert to wild type by homologous recombination. All chemicals were positive in both assays except for 3-hydroxy-trans-stilbene, which was negative in both. HU, resveratrol, and 4-hydroxy-trans-stilbene were scavengers of the tyrosyl free radical of the R2 subunit of mammalian ribonucleotide reductase. Tyrosyl free radical scavengers disturb normal DNA replication, causing replication fork arrest. Mitomycin C is a DNA cross-linking agent that also causes replication fork arrest. The present study suggests that replication fork arrest, which is similar to the early phases of HR, leads to a high frequency of recombination, resulting in SCEs. The findings show that SCE may be mediated by HR.

  • 16.
    Ström, Cecilia E.
    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.
    Uhlén, Mathias
    Al-Khalili Szigyarto, Cristina
    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.
    Poly (ADP-ribose) polymerase (PARP) is not involved in base excision repair but PARP inhibition traps a single-strand intermediate2011In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 39, no 8, p. 3166-3175Article in journal (Refereed)
    Abstract [en]

    Base excision repair (BER) represents the most important repair pathway of endogenous DNA lesions. Initially, a base damage is recognized, excised and a DNA single-strand break (SSB) intermediate forms. The SSB is then ligated, a process that employs proteins also involved in SSB repair, e.g. XRCC1, Ligase III and possibly PARP1. Here, we confirm the role of XRCC1 and PARP in direct SSB repair. Interestingly, we uncover a synthetic lethality between XRCC1 deficiency and PARP inhibition. We also treated cells with alkylating agent dimethyl sulfate (DMS) and monitored the SSB intermediates formed during BER. DMS-induced SSBs were quickly repaired in wild-type cells; while a rapid accumulation of SSBs was observed in cells where post-incision repair was blocked by a PARP inhibitor or by XRCC1 deficiency (EM9 cells). Interestingly, DMS-induced SSBs did not accumulate in PARP1 siRNA depleted cells, demonstrating that PARP1 is not required for efficient completion of BER. Based on these results we suggest no immediate role for PARP1 in BER, but that PARP inhibitors trap PARP on the SSB intermediate formed during BER. Unexpectedly, addition of PARP inhibitor 2 h after DMS treatment still increased SSB levels indicating ongoing repair even at this late time point.

  • 17.
    Ström, Cecilia E.
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Mortusewicz, Oliver
    Finch, David
    Parsons, Jason L.
    Lagerqvist, Anne
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    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.
    Erixon, Klaus
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Dianov, Grigory L.
    Helleday, Thomas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    CK2 phosphorylation of XRCC1 facilitates dissociation from DNA and single-strand break formation during base excision repair2011In: DNA Repair, ISSN 1568-7864, E-ISSN 1568-7856, Vol. 10, no 9, p. 961-969Article in journal (Refereed)
    Abstract [en]

    CK2 phosphorylates the scaffold protein XRCC1, which is required for efficient DNA single-strand break (SSB) repair. Here, we express an XRCC1 protein (XRCC1(ckm)) that cannot be phosphorylated by CK2 in XRCC1 mutated EM9 cells and show that the role of this post-translational modification gives distinct phenotypes in SSB repair and base excision repair (BER). Interestingly, we find that fewer SSBs are formed during BER after treatment with the allcylating agent dimethyl sulfate (DMS) in EM9 cells expressing XRCC1(ckm) (CKM cells) or following inhibition with the CK2 inhibitor 2-dimethylamino-4,5,6,7tetrabromo-1H-benzimidazole (DMAT). We also show that XRCC1(ckm) protein has a higher affinity for DNA than wild type XRCC1 protein and resides in an immobile fraction on DNA, in particular after damage. We propose a model whereby the increased affinity for DNA sequesters XRCC1(ckm) and the repair enzymes associated with it, at the repair site, which retards kinetics of BER. In conclusion, our results indicate that phosphorylation of XRCC1 by CK2 facilitates the BER incision step, likely by promoting.

  • 18.
    Ström, Cecilia
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Lagerqvist, Anne
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Finch, David
    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.
    Erixon, Klaus
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Dianov, Grigory
    Helleday, Thomas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    CK2 phosphorylation of XRCC1 facilitate single-strand break formation during base excision repairManuscript (preprint) (Other academic)
    Abstract [en]

    Casein kinase 2 (CK2) phosphorylates the scaffold protein XRCC1, which is required for efficient DNA single-strand break (SSB) repair. Here, we express a XRCC1 protein (XRCC1ckm) that cannot be phosphorylated by CK2 in XRCC1 mutated EM9 cells and show that the role of this post-translational modification in SSB repair is distinct from its role in base excision repair (BER). Interestingly, we find that fewer SSBs are formed during BER after treatment with the alkylating agent dimethyl sulfate (DMS) in EM9 cells expressing XRCC1ckm (CKM cells) or following inhibition with CK2 inhibitor 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT). We also show that XRCC1ckm protein has a higher affinity for nicked DNA substrate than wild type XRCC1 protein and we propose a model whereby the increased affinity for DNA sequesters XRCC1ckm and the repair enzymes associated with it, at the repair site. In conclusion, our results indicate that CK2-phosphorylations of XRCC1 affect the kinetics of SSB repair and BER differentially, and that the modifications on XRCC1 facilitate the BER incision step.

  • 19. Syljuåsen, Randi G
    et al.
    Sørensen, Claus Storgaard
    Hansen, Lasse Tengbjerg
    Fugger, Kasper
    Lundin, Cecilia
    Stockholm University.
    Johansson, Fredrik
    Stockholm University.
    Helleday, Thomas
    Stockholm University.
    Sehested, Maxwell
    Lukas, Jiri
    Bartek, Jiri
    Inhibition of human Chk1 causes increased initiation of DNA replication, phosphorylation of ATR targets, and DNA breakage.2005In: Mol Cell Biol, ISSN 0270-7306, Vol. 25, no 9, p. 3553-62Article in journal (Refereed)
1 - 19 of 19
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