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Uncoupling of RAD51 focus formation and cell survival after replication fork stalling in RAD51D null CHO cells
Lawrence Livermore Natl. Laboratory, Biosciences and Biotechnology Division.
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology. (Thomas Helleday)
Washington State University, School of Molecular biosciences.
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology. (Thomas Helleday)
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(English)Article in journal (Refereed) Submitted
Abstract [en]

In vertebrate cells the five RAD51 paralogs (XRCC2/3, RAD51B/C/D) enhance the efficiency of homologous reocmbination repair (HRR). Stalling and breakage of DNA replication forks is a common event in the large genomes of higher eukaryotes. When cells are exposed to agents that arrest DNA replicaiton, such as hydroxyurea or aphidicolin, fork breakage can lead to chromosomal aberrations and cell killing. We assessed the contribution of the HRR protein RAD51D in resistance to killing by replication-associated DSBs. In repsonse to hydroxyurea, the isogenic rad51d null CHO mutant fails to show any indication of HRR initiation, as assessed by induction of RAD51 foci, as expected. Surprisingly, these cells have normal resistance to killing by replication inhibition from either hydroxyurea or aphidicolin, but show the expected sensitivity to camptothecin, which also generates replication-dependent DSBs. In contrast, we confirm that the V79 xrcc2 mutant does show increased sensitivity to hydroxyurea under some conditinos, which was correlated to its attenuated RAD51 focus response. In response to a PARP1 inhibitor PARP1, rad51d cells, like other HRR mutants, show exquisite sensitivity (>1000 fold), which is also associated with defective RAD51 focus formation. Thus, rad51d cells are broadly deficient in RAD51 focus formation in response to various agents, but this defect is not invariably associated with increased sensitivity. Our results indicate that RAD51 paralogs do not contribute equally to cellular resistance of inhibitors of DNA replication, and that the RAD51 foci associated with replication inhibition may not be a reliable indicator of cellular resistance to such agents.

National Category
Natural Sciences
URN: urn:nbn:se:su:diva-56696OAI: diva2:412234
Available from: 2011-04-21 Created: 2011-04-21 Last updated: 2011-04-27Bibliographically approved
In thesis
1. Replication Fork Stability in Mammalian Cells
Open this publication in new window or tab >>Replication Fork Stability in Mammalian Cells
2011 (English)Doctoral 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.

Place, publisher, year, edition, pages
Stockholm: Department of Genetics, Microbiology and Toxicology, Stockholm University, 2011. 77 p.
replication fork progression, replication fork stability, re-priming, DNA damage signalling
National Category
Natural Sciences
Research subject
Molecular Genetics
urn:nbn:se:su:diva-56697 (URN)978-91-7447-270-7 (ISBN)
Public defence
2011-05-26, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 1: Submitted. Paper 2: Submitted. Paper 3: Manuscript. Paper 5: Submitted.Available from: 2011-05-04 Created: 2011-04-21 Last updated: 2011-06-21Bibliographically approved

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