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Replication Fork Stability in Mammalian Cells
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology. (Thomas Helleday)
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.
Keyword [en]
replication fork progression, replication fork stability, re-priming, DNA damage signalling
National Category
Natural Sciences
Research subject
Molecular Genetics
Identifiers
URN: urn:nbn:se:su:diva-56697ISBN: 978-91-7447-270-7OAI: oai:DiVA.org:su-56697DiVA: diva2:412246
Public defence
2011-05-26, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
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
List of papers
1. UV stalled replication forks restart by re-priming in human fibroblasts
Open this publication in new window or tab >>UV stalled replication forks restart by re-priming in human fibroblasts
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2011 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 39, no 16, 7049-7057 p.Article in journal (Refereed) Published
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.

National Category
Natural Sciences
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:su:diva-56691 (URN)10.1093/nar/gkr420 (DOI)000294556800024 ()
Available from: 2011-04-21 Created: 2011-04-21 Last updated: 2012-01-03Bibliographically approved
2. CHK1 activity is required for replication fork elongation but not stabilisation after UV irradiation
Open this publication in new window or tab >>CHK1 activity is required for replication fork elongation but not stabilisation after UV irradiation
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(English)Article in journal (Refereed) Submitted
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. 

National Category
Natural Sciences
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:su:diva-56694 (URN)
Available from: 2011-04-21 Created: 2011-04-21 Last updated: 2011-04-27Bibliographically approved
3. UV-induced replication fork collapse in DNA polymerase η deficient cells is independent of the MUS81 endonuclease
Open this publication in new window or tab >>UV-induced replication fork collapse in DNA polymerase η deficient cells is independent of the MUS81 endonuclease
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(English)Manuscript (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.

National Category
Natural Sciences
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:su:diva-56692 (URN)
Available from: 2011-04-21 Created: 2011-04-21 Last updated: 2011-04-27Bibliographically approved
4. The histone methyltransferase SET8 is required for S-phase progression
Open this publication in new window or tab >>The histone methyltransferase SET8 is required for S-phase progression
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2007 (English)In: Journal of Cell Biology, ISSN 0021-9525, E-ISSN 1540-8140, Vol. 179, no 7, 1337-1345 p.Article in journal (Refereed) Published
Abstract [en]

Chromatin structure and function is influenced by histone posttranslational modifications. SET8 (also known as PR-Set7 and SETD8) is a histone methyltransferase that monomethylates histone H4-K20. However, a function for SET8 in mammalian cell proliferation has not been determined. We show that small interfering RNA inhibition of SET8 expression leads to decreased cell proliferation and accumulation of cells in S phase. This is accompanied by DNA double-strand break (DSB) induction and recruitment of the DNA repair proteins replication protein A, Rad51, and 53BP1 to damaged regions. SET8 depletion causes DNA damage specifically during replication, which induces a Chk1-mediated S-phase checkpoint. Furthermore, we find that SET8 interacts with proliferating cell nuclear antigen through a conserved motif, and SET8 is required for DNA replication fork progression. Finally, codepletion of Rad51, an improtant homologous recombination repair protein, abrogates the DNA damage after SET8 depletion. Overall, we show that SET8 is essential for genomic stability in mammalian cells and that decreased expression of SET8 results in DNA damage and Chk1-dependent S-phase arrest.

National Category
Natural Sciences
Identifiers
urn:nbn:se:su:diva-56695 (URN)10.1083/jcb.200706150 (DOI)
Available from: 2011-04-21 Created: 2011-04-21 Last updated: 2011-04-27Bibliographically approved
5. Uncoupling of RAD51 focus formation and cell survival after replication fork stalling in RAD51D null CHO cells
Open this publication in new window or tab >>Uncoupling of RAD51 focus formation and cell survival after replication fork stalling in RAD51D null CHO cells
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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
Identifiers
urn:nbn:se:su:diva-56696 (URN)
Available from: 2011-04-21 Created: 2011-04-21 Last updated: 2011-04-27Bibliographically approved

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