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Interplay between Transcription and Homologous Recombination in the Presence of DNA Damage
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology. (Thomas Helleday's research group)
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The biochemical processes of DNA repair, replication and recombination compete for the same substrate, the DNA molecule. This competition is natural, as each process requires the same template. In order to resolve possible conflicts between these processes, when they take place on a particular stretch of DNA, certain crosstalk is expected. The complexity is additionally increased by the existence of another DNA dependent process, which occurs in all phases of the cell cycle: transcription.

This thesis aims to investigate the link between transcription inhibition and homologous recombination, especially in the context of UV-induced DNA damage. The results show that the transcription inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) induces homologous recombination. The DNA damage caused by UVC irradiation is repaired mainly via nucleotide excision repair, however, it is also known to trigger recombinational repair. In the presence of UV-induced damage, transcription inhibition by DRB potentiates the induction of homologous recombination as a necessary mechanism of cell survival.

The thesis also focuses on the toxicity mechanisms of the chemotherapeutic compound 6-thioguanine (6TG). The work in the thesis suggests application of 6TG as a treatment for hereditary forms of breast cancer, with genetically altered BRCA1 or BRCA2 functions. Most importantly, the treatment with 6TG is applicable to breast cancers, which have developed resistance to another class of chemotherapeutic drugs, poly-(ADP-ribose) polymerase (PARP) inhibitors. Repair of the DNA damage induced by 6TG treatment is investigated further with a particular focus the pathway of DNA damage avoidance involving DNA polymerase η. The function of DNA polymerase η seems to be an important factor for the outcome of DNA repair after 6TG exposure. The deficiency of DNA polymerase η is also investigated in connection with normal replication and the repair of UV-induced DNA damage.

In summary, the work in the thesis sheds more light onto the fundamental connections between DNA replication, recombination, transcription, repair and damage avoidance. On a more practical side, the information obtained about these fundamental connections is used to suggest a possible therapy for several forms of breast cancer.

Place, publisher, year, edition, pages
Stockholm: Department of Genetics, Microbiology and Toxicology, Stockholm University , 2011. , 70 p.
Keyword [en]
homologous recombination, transcription, DRB, UVC, 6-thioguanine, mammalian cells, BRCA1, BRCA2, PARP inhibitors, XP-V
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Genetics
Identifiers
URN: urn:nbn:se:su:diva-54736ISBN: 978-91-7447-217-2 (print)OAI: oai:DiVA.org:su-54736DiVA: diva2:397404
Public defence
2011-03-18, De Geersalen, Geovetenskapens hus, Svante Arrhenius väg 14, Stockholm, 13:00 (English)
Opponent
Supervisors
Note
At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 4: Manuscript. Paper 5: Manuscript.Available from: 2011-02-24 Created: 2011-02-14 Last updated: 2011-02-16Bibliographically approved
List of papers
1. Transcription inhibition by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) causes DNA damage and triggers homologous recombination repair in mammalian cells
Open this publication in new window or tab >>Transcription inhibition by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) causes DNA damage and triggers homologous recombination repair in mammalian cells
2011 (English)In: Mutation research, ISSN 0027-5107, E-ISSN 1873-135X, Vol. 706, no 1-2, 1-6 p.Article in journal (Refereed) Published
Abstract [en]

Transcription, replication and homologous recombination are intrinsically connected and it is well established that an increase of transcription is associated with an increase in homologous recombination. Here, we have studied how homologous recombination is affected during transcription inhibition by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), a compound that prevents activating phosphorylations of the RNA Pol II C-terminal domain. We identify that DRB triggers an increase in homologous recombination within the hprt gene as well as increasing RAD51 foci formation in mammalian cells. Furthermore, we find that DRB-induced transcriptional stress is associated with formation of the nuclear foci of the phosphorylated form of H2AX (γH2AX). We accounted that about 72% of RAD51 foci co-localized with the observed γH2AX foci. Interestingly, we find that XRCC3 mutated, homologous recombination defective cells are hypersensitive to the toxic effect of DRB and fail to form RAD51 foci. In conclusion, we show that DRB-induced transcription inhibition is associated with the formation of a lesion that triggers RAD51-dependent homologous recombination repair, required for survival under transcriptional stress.

Keyword
Homologous recombination, DRB, Transcription, DNA damage repair, Mammalian cells
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:su:diva-54714 (URN)10.1016/j.mrfmmm.2010.10.012 (DOI)000287123600001 ()
Available from: 2011-02-14 Created: 2011-02-13 Last updated: 2017-12-11Bibliographically approved
2. Transcription inhibition by DRB potentiates recombinational repair of UVC lesions in mammalian cells
Open this publication in new window or tab >>Transcription inhibition by DRB potentiates recombinational repair of UVC lesions in mammalian cells
(English)Manuscript (preprint) (Other academic)
Keyword
Transcription, homologous recombination, DRB, UVC
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:su:diva-54731 (URN)
Available from: 2011-02-14 Created: 2011-02-14 Last updated: 2011-02-16Bibliographically approved
3. 6-Thioguanine Selectively Kills BRCA2-Defective Tumors and Overcomes PARP Inhibitor Resistance
Open this publication in new window or tab >>6-Thioguanine Selectively Kills BRCA2-Defective Tumors and Overcomes PARP Inhibitor Resistance
Show others...
2010 (English)In: Cancer Research, ISSN 0008-5472, E-ISSN 1538-7445, Vol. 70, no 15, 6268-6276 p.Article in journal (Refereed) Published
Abstract [en]

Familial breast and ovarian cancers are often defective in homologous recombination (HR) due to mutations in the BRCA1 or BRCA2 genes. Cisplatin chemotherapy or poly(ADP-ribose) polymerase (PARP) inhibitors were tested for these tumors in clinical trials. In a screen for novel drugs that selectively kill BRCA2-defective cells, we identified 6-thioguanine (6TG), which induces DNA double-strand breaks (DSB) that are repaired by HR. Furthermore, we show that 6TG is as efficient as a PARP inhibitor in selectively killing BRCA2-defective tumors in a xenograft model. Spontaneous BRCA1-defective mammary tumors gain resistance to PARP inhibitors through increased P-glycoprotein expression. Here, we show that 6TG efficiently kills such BRCA1-defective PARP inhibitor-resistant tumors. We also show that 6TG could kill cells and tumors that have gained resistance to PARP inhibitors or cisplatin through genetic reversion of the BRCA2 gene. Although HR is reactivated in PARP inhibitor-resistant BRCA2-defective cells, it is not fully restored for the repair of 6TG-induced lesions. This is likely to be due to several recombinogenic lesions being formed after 6TG. We show that BRCA2 is also required for survival from mismatch repair-independent lesions formed by 6TG, which do not include DSBs. This suggests that HR is involved in the repair of 6TG-induced DSBs as well as mismatch repair-independent 6TG-induced DNA lesion. Altogether, our data show that 6TG efficiently kills BRCA2-defective tumors and suggest that 6TG may be effective in the treatment of advanced tumors that have developed resistance to PARP inhibitors or platinum-based chemotherapy. Cancer Res; 70(15); 6268-76. (C) 2010 AACR.

National Category
Biochemistry and Molecular Biology
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:su:diva-49721 (URN)10.1158/0008-5472.CAN-09-3416 (DOI)000280557500017 ()
Note

authorCount :20

Available from: 2010-12-17 Created: 2010-12-17 Last updated: 2017-12-11Bibliographically approved
4. UVC–stalled replication forks readily collapse into DNA double-strand breaksin the absence of DNA polymerase η and independently of Mus81 in humancells
Open this publication in new window or tab >>UVC–stalled replication forks readily collapse into DNA double-strand breaksin the absence of DNA polymerase η and independently of Mus81 in humancells
Show others...
(English)Manuscript (preprint) (Other academic)
Keyword
Polη, translesion synthesis, homologous recombination, ultraviolet
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:su:diva-54732 (URN)
Available from: 2011-02-14 Created: 2011-02-14 Last updated: 2011-02-16Bibliographically approved
5. Polymerase η proficiency sensitises cells to 6-thioguanine
Open this publication in new window or tab >>Polymerase η proficiency sensitises cells to 6-thioguanine
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Severe photosensitivity of the skin and predisposition to cancer development are two important features whichcharacterising a genetic syndrome known as Xeroderma pigmentosum. An interesting class of patients has beendescribed, characterised by a proficiency in nucleotide excision repair and a defect in the DNA damage avoidancepathways. This class is termed Xeroderma pigmentosum variant (XP-V) and is known to be caused by a deficiencyin the function of the specific DNA Polymerase η. Cells derived from XP-V patients are sensitivie not only to UVlight but also to crosslinkers such as cisplatin, and Polη overexpression is potentially relevant to development ofcisplatin resistance. In this paper we investigate the importance of the Polη status in the response to treatment withthe chemotherapeutic agent 6-thioguanine (6TG). Our results show that Polη deficient cells are more resistant totreatment with 6TG in comparison to Polη complemented cells. This is in contrast to the typical UV sensitivity ofPolη deficient cells, which is confirmed in the same cells. We also show that 6TG has a growth retardation effect,regardless of the Polη status. There were no DNA double-strand breaks detected in a short period after theexposure to 6TG in physiologically relevant doses, although a DNA damage response was observed in high doses.It was previously demonstrated that 6TG can be used to kill cisplatin resistant cells and these data may indicateone potential explanation.

Keyword
Polη, 6-thioguanine, XP-V
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:su:diva-54733 (URN)
Available from: 2011-02-14 Created: 2011-02-14 Last updated: 2011-02-16Bibliographically approved

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