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UV-induced replication fork collapse in DNA polymerase η deficient cells is independent of the MUS81 endonuclease
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology. (Thomas Helleday)
Joint Institute for Nuclear Research, Dubna, Russia. (Andrzej Wojcik)
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology. (Thomas Helleday)
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology. (Thomas Helleday)
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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: urn:nbn:se:su:diva-56692OAI: oai:DiVA.org:su-56692DiVA: diva2:412225
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.
Keyword
replication fork progression, replication fork stability, re-priming, DNA damage signalling
National Category
Natural Sciences
Research subject
Molecular Genetics
Identifiers
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)
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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

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Elvers, IngegerdJohansson, FredrikSchultz, NiklasWojcik, AndrzejHelleday, Thomas
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