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CHK1 activity is required for replication fork elongation but not stabilisation after UV irradiation
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)
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)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: urn:nbn:se:su:diva-56694OAI: oai:DiVA.org:su-56694DiVA: diva2:412228
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, FredrikLagerqvist, AnneStoimenov, IvayloErixon, KlausHelleday, Thomas
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