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Homologous recombination at replication forks in mammalian cells
Stockholm University, Faculty of Social Sciences, Department of Applied Communications Science - GI and IHR.
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Accumulating evidence indicates that homologous recombination plays an important role in mammalian cells, primarily in the reactivation of stalled replication forks. During a normal round of replication, stalling of the replication fork occurs frequently when a nick or a lesion, which cannot be by-passed, is encountered. Progression of the replication fork can also be hindered by a protein bound to or secondary structures in the DNA. It is vital for the cell to restart stalled replication forks efficiently since nucleases might otherwise damage the unprotected DNA and since failure to reinitiate replication leads ultimately to cell death. The mechanism by which homologous recombination reactivates stalled replication forks and the substrates that trigger this response in mammalian cells have not yet been elucidated.

The present thesis focuses on the function of homologous recombination as well as on the mechanism for induction of this process in mammalian cells. Our major interest in this context was the repair of lesions encountered during replication and that interfere with replication fork progression. The role of the RAD51 protein in this process was also examined. The main experimental system we employed consisted of Chinese hamster cell lines deficient in different DNA repair pathways, as well as the V79 Chinese hamster SPD8 cell line, which contains an endogenous locus at which homologous recombination events can be monitored.

Our findings demonstrate that homologous recombination can be employed by mammalian cells to repair several different types of lesions encountered during replication. The mechanism underlying such homologous recombinational repair appears to depend on the type of lesion involved. A replication fork encountering a single-strand break was shown to collapse into a double-strand break and subsequently be repaired by homologous recombination involving a mechanism referred to as break-induced replication. A stalled replication fork was shown to induce a substrate for homologous recombination both with and without the induction of a double-strand break. This suggests that homologous recombination could repair lesions other than double-strand breaks during replication. Non-homologous end-joining was shown to play only a minor role in the repair of DNA damage associated with replication, being employed only in situations when the replication fork is processed into a double-strand break.

Experiments concerning the role of RAD51 in repair of lesions encountered during replication, employing a cell line that overexpresses this protein, indicated that RAD51 is rate limiting for the repair of DNA damage associated with the replication fork. The level of RAD51 was also measured in lung cancer cells and found to determine both the survival and the level of double-strand breaks formed in response to etoposide treatment, suggesting RAD51 to be involved in the resistance of tumours to chemotherapeutic drugs.

In summary, our findings demonstrate that RAD51-dependent homologous recombination is an important repair pathway during replication in mammalian cells.

Place, publisher, year, edition, pages
Stockholm: Institutionen för genetik, mikrobiologi och toxikologi , 2004. , 64 p.
National Category
Genetics
Identifiers
URN: urn:nbn:se:su:diva-207ISBN: 91-7265-922-X (print)OAI: oai:DiVA.org:su-207DiVA: diva2:190951
Public defence
2004-09-10, Nordenskiöldsalen, Geovetenskapens hus, Svante Arrhenius väg 8 C, Stockholm, 13:00
Opponent
Supervisors
Available from: 2004-08-20 Created: 2004-08-20Bibliographically approved
List of papers
1. DNA Double-strand Breaks Associated with Replication Forks are Predominantly Repaired by Homologous Recombination Involving an Exchange Mechanism in Mammalian Cells
Open this publication in new window or tab >>DNA Double-strand Breaks Associated with Replication Forks are Predominantly Repaired by Homologous Recombination Involving an Exchange Mechanism in Mammalian Cells
2001 (English)In: Journal of Molecular Biology, ISSN 0022-2836, Vol. 307, no 5, 1235-45 p.Article in journal (Refereed) Published
Abstract [en]

DNA double-strand breaks (DSB) represent a major disruption in the integrity of the genome. DSB can be generated when a replication fork encounters a DNA lesion. Recombinational repair is known to resolve such replication fork-associated DSB, but the molecular mechanism of this repair process is poorly understood in mammalian cells. In the present study, we investigated the molecular mechanism by which recombination resolves camptothecin (CPT)-induced DSB at DNA replication forks. The frequency of homologous recombination (HR) was measured using V79/SPD8 cells which contain a duplication in the endogenous hprt gene that is resolved by HR. We demonstrate that DSB associated with replication forks induce HR at the hprt gene in early S phase. Further analysis revealed that these HR events involve an exchange mechanism. Both the irs1SF and V3-3 cell lines, which are deficient in HR and non-homologous end joining (NHEJ), respectively, were found to be more sensitive than wild-type cells to DSB associated with replication forks. The irs1SF cell line was more sensitive in this respect than V3-3 cells, an observation consistent with the hypothesis that DSB associated with replication forks are repaired primarily by HR. The frequency of formation of DSB associated with replication forks was not affected in HR and NHEJ deficient cells, indicating that the loss of repair, rather than the formation of DSB associated with replication forks is responsible for the increased sensitivity of the mutant strains. We propose that the presence of DSB associated with replication forks rapidly induces HR via an exchange mechanism and that HR plays a more prominent role in the repair of such DSB than does NHEJ

Place, publisher, year, edition, pages
Elsevier, 2001
Keyword
replication fork; DNA double-strand break; homologous recombination; exchange mechanism; hypoxanthine-guanine phosphoribosyltransferase
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-23255 (URN)10.1006/jmbi.2001.4564 (DOI)
Note
Part of urn:nbn:se:su:diva-207Available from: 2004-08-20 Created: 2004-08-20 Last updated: 2010-01-07Bibliographically approved
2. Different Roles for Nonhomologous End Joining and Homologous Recombination following Replication Arrest in Mammalian Cells
Open this publication in new window or tab >>Different Roles for Nonhomologous End Joining and Homologous Recombination following Replication Arrest in Mammalian Cells
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2002 (English)In: Molecular and Cellular Biology, ISSN 0270-7306, Vol. 22, no 16, 5869-78 p.Article in journal (Refereed) Published
Abstract [en]

Homologous recombination (HR) and nonhomologous end joining (NHEJ) play overlapping roles in repair of DNA double-strand breaks (DSBs) generated during the S phase of the cell cycle. Here, we characterized the involvement of HR and NHEJ in the rescue of DNA replication forks arrested or slowed by treatment of hamster cells with hydroxyurea or thymidine. We show that the arrest of replication with hydroxyurea generates DNA fragmentation as a consequence of the formation of DSBs at newly replicated DNA. Both HR and NHEJ protected cells from the lethal effects of hydroxyurea, and this agent also increased the frequency of recombination mediated by both homologous and nonhomologous exchanges. Thymidine induced a less stringent arrest of replication and did not generate detectable DSBs. HR alone rescued cells from the lethal effects of thymidine. Furthermore, thymidine increased the frequency of DNA exchange mediated solely by HR in the absence of detectable DSBs. Our data suggest that both NHEJ and HR are involved in repair of arrested replication forks that include a DSB, while HR alone is required for the repair of slowed replication forks in the absence of detectable DSBs.

Place, publisher, year, edition, pages
American Society of Microbiology, 2002
National Category
Biological Sciences
Research subject
Cellbiology
Identifiers
urn:nbn:se:su:diva-23256 (URN)10.1128/MCB.22.16.5869-5878.2002 (DOI)
Note
Part of urn:nbn:se:su:diva-207Available from: 2004-08-20 Created: 2004-08-20 Last updated: 2010-01-07Bibliographically approved
3. Overexpression of Cyclin E Does not Affect Homologous Recombination in Chinese Hamster Cells
Open this publication in new window or tab >>Overexpression of Cyclin E Does not Affect Homologous Recombination in Chinese Hamster Cells
2003 (English)In: Biochemical and Biophysical Research Communications, ISSN 0006-291X, Vol. 296, no 2, 363-7 p.Article in journal (Refereed) Published
Abstract [en]

Overexpressed cyclin E in tumours is a prognosticator for poor patient outcome. Cells that overexpress cyclin E have been shown to be impaired in S-phase progression and exhibit genetic instability that may drive this subset of cancers. However, the origin for genetic instability caused by cyclin E overexpression is unknown. Homologous recombination plays an important role in S-phase progression and is also regulated by the same proteins that regulate cyclin E-associated kinase activity, i.e., p53 and p21. To test the hypothesis that overexpressed cyclin E causes genetic instability through homologous recombination, we investigated the effect of cyclin E overexpression on homologous recombination in the hprt gene in a Chinese hamster cell line. Although cyclin E overexpression shortened the G1 phase in the cell cycle as expected, we could see no change in neither spontaneous nor etoposide-induced recombination. Also, overexpression of cyclin E did not affect the repair of DNA double-strand breaks and failed to potentiate the cytotoxic effects of etoposide. Our data suggest that genetic instability caused by overexpression of cyclin E is not mediated by aberrant homologous recombination

Place, publisher, year, edition, pages
Elsevier, 2003
Keyword
Cyclin E; Homologous recombination; Etoposide; Genetic instability; Hypoxanthine-guanine phosphoribosyltransferase gene
National Category
Biological Sciences
Research subject
Cellbiology
Identifiers
urn:nbn:se:su:diva-23257 (URN)10.1016/S0006-291X(02)00889-6 (DOI)
Note
Part of urn:nbn:se:su:diva-207Available from: 2004-08-20 Created: 2004-08-20 Last updated: 2010-01-07Bibliographically approved
4. RAD51 is Involved in Repair of Damage Associated with DNA Replication in Mammalian Cells
Open this publication in new window or tab >>RAD51 is Involved in Repair of Damage Associated with DNA Replication in Mammalian Cells
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2003 (English)In: Journal of Molecular Biology, ISSN 0022-2836, Vol. 328, no 3, 521-35 p.Article in journal (Refereed) Published
Abstract [en]

The RAD51 protein, a eukaryotic homologue of the Escherichia coli RecA protein, plays an important role in the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) in mammalian cells. Recent findings suggest that HR may be important in repair following replication arrest in mammalian cells. Here, we have investigated the role of RAD51 in the repair of different types of damage induced during DNA replication with etoposide, hydroxyurea or thymidine. We show that etoposide induces DSBs at newly replicated DNA more frequently than γ-rays, and that these DSBs are different from those induced by hydroxyurea. No DSB was found following treatment with thymidine. Although these compounds appear to induce different DNA lesions during DNA replication, we show that a cell line overexpressing RAD51 is resistant to all of them, indicating that RAD51 is involved in repair of a wide range of DNA lesions during DNA replication. We observe fewer etoposide-induced DSBs in RAD51-overexpressing cells and that HR repair of etoposide-induced DSBs is faster. Finally, we show that induced long-tract HR in the hprt gene is suppressed in RAD51-overexpressing cells, although global HR appears not to be suppressed. This suggests that overexpression of RAD51 prevents long-tract HR occurring during DNA replication. We discuss our results in light of recent models suggested for HR at stalled replication forks.

Place, publisher, year, edition, pages
Elsevier, 2003
Keyword
RAD51; mammalian cells; homologous recombination; stalled DNA replication forks; DNA double-strand breaks
National Category
Biological Sciences
Research subject
Cellbiology
Identifiers
urn:nbn:se:su:diva-23258 (URN)10.1016/S0022-2836(03)00313-9 (DOI)
Note
Part of urn:nbn:se:su:diva-207Available from: 2004-08-20 Created: 2004-08-20 Last updated: 2010-01-07Bibliographically approved
5. The Role of RAD51 in Etoposide (VP16) Resistance in Small Cell Lung Cancer
Open this publication in new window or tab >>The Role of RAD51 in Etoposide (VP16) Resistance in Small Cell Lung Cancer
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2003 (English)In: International Journal of Cancer, ISSN 0020-7136, Vol. 105, no 4, 472-9 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-23259 (URN)
Note
Part of urn:nbn:se:su:diva-207Available from: 2004-08-20 Created: 2004-08-20 Last updated: 2010-01-29Bibliographically approved
6. DNA Repair Rate and Etoposide (VP16) Resistance of Tumor Cell Subpopulations derived from a Single Human Small Cell Lung Cancer
Open this publication in new window or tab >>DNA Repair Rate and Etoposide (VP16) Resistance of Tumor Cell Subpopulations derived from a Single Human Small Cell Lung Cancer
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2003 (English)In: Lung Cancer, ISSN 0169-5002, Vol. 40, no 2, 157-64 p.Article in journal (Refereed) Published
Abstract [en]

Two human small cell lung cancer (SCLC) subpopulations, CPH 54A, and CPH 54B, established from the same patient tumor by in vitro cloning, were investigated. The tumor was classified as intermediate-type SCLC. The cellular sensitivity to ionizing radiation (IR) was previously determined in the two sublines both in vivo and in vitro. Here we measured the etoposide (VP16) sensitivity together with the induction and repair of VP16- and IR-induced DNA double-strand breaks (DSBs). The two subpopulations were found to differ significantly in sensitivity to VP16, with the radioresistant 54B subline also being VP16 resistant. In order to explain the VP16 resistant phenotype several mechanisms where considered. The p53 status, P-glycoprotein, MRP, topoisomerase IIα, and Mre11 protein levels, as well as growth kinetics, provided no explanations of the observed VP16 resistance. In contrast, a significant difference in repair of both VP16- and IR-induced DSBs, together with a difference in the levels of the DSB repair proteins DNA-dependent protein kinase (DNA-PKcs) and RAD51 was observed. The VP16- and radioresistant 54B subline exhibited a pronounced higher repair rate of DSBs and higher protein levels of both DNA-PKcs and RAD51 compared with the sensitive 54A subline. We suggest, that different DSB repair rates among tumor cell subpopulations of individual SCLC tumors may be a major determinant for the variation in clinical treatment effect observed in human SCLC tumors of identical histological subtype.

Place, publisher, year, edition, pages
Elsevier, 2003
Keyword
DNA-PKcs; RAD51; DNA double-strand break repair; VP16; Etoposide; Ionizing radiation; Drug resistance; Small cell lung cancer
National Category
Biological Sciences
Research subject
Cellbiology
Identifiers
urn:nbn:se:su:diva-23260 (URN)10.1016/S0169-5002(03)00026-6 (DOI)
Note
Part of urn:nbn:se:su:diva-207Available from: 2004-08-20 Created: 2004-08-20 Last updated: 2010-01-07Bibliographically approved
7. Alkylating DNA Lesions Associated with Replication are Repaired by Homologous Recombination in Mammalian Cells
Open this publication in new window or tab >>Alkylating DNA Lesions Associated with Replication are Repaired by Homologous Recombination in Mammalian Cells
Manuscript (Other academic)
Identifiers
urn:nbn:se:su:diva-23261 (URN)
Note
Part of urn:nbn:se:su:diva-207Available from: 2004-08-20 Created: 2004-08-20 Last updated: 2010-01-13Bibliographically approved

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  • apa
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  • nn-NB
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Output format
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