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Regulators of cyclin-dependent kinases are crucial for maintaining genome integrity in S phase
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
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2010 (English)In: Journal of Cell Biology, ISSN 0021-9525, E-ISSN 1540-8140, Vol. 188, no 5, 629-638 p.Article in journal (Refereed) Published
Abstract [en]

Maintenance of genome integrity is of critical importance to cells. To identify key regulators of genomic integrity, we screened a human cell line with a kinome small interfering RNA library. WEE1, a major regulator of mitotic entry, and CHK1 were among the genes identified. Both kinases are important negative regulators of CDK1 and -2. Strikingly, WEE1 depletion rapidly induced DNA damage in S phase in newly replicated DNA, which was accompanied by a marked increase in single-stranded DNA. This DNA damage is dependent on CDK1 and -2 as well as the replication proteins MCM2 and CDT1 but not CDC25A. Conversely, DNA damage after CHK1 inhibition is highly dependent on CDC25A. Furthermore, the inferior proliferation of CHK1-depleted cells is improved substantially by codepletion of CDC25A. We conclude that the mitotic kinase WEE1 and CHK1 jointly maintain balanced cellular control of Cdk activity during normal DNA replication, which is crucial to prevent the generation of harmful DNA lesions during replication.

Place, publisher, year, edition, pages
2010. Vol. 188, no 5, 629-638 p.
National Category
Natural Sciences
URN: urn:nbn:se:su:diva-50311DOI: 10.1083/jcb.200905059ISI: 000275331800004OAI: diva2:380777
authorCount :10Available from: 2010-12-22 Created: 2010-12-22 Last updated: 2011-02-28Bibliographically approved
In thesis
1. Replication Dynamics in the DNA Damage Response
Open this publication in new window or tab >>Replication Dynamics in the DNA Damage Response
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Faithful DNA replication is essential and the induction of replication stress may have profound effects on genomic integrity. This is demonstrated by the formation of DNA double strand breaks (DSBs), considered to be the most toxic DNA lesions, at stalled replication forks. Homologous recombination (HR) has been shown to be involved in the replication stress response and has been suggested for stabilisation, restart and repair of stalled replication forks. However, the HR mechanisms induced by replication stress are still, to a major part, unknown. The present thesis focuses on investigating replication patterns following the induction of replication stress. Further, the consequences of stressed replication are studied by detection of DSB formation and characterisation of HR in mammalian cells.

Here, we have identified WEE1, a regulator of mitotic entry, as a factor required to maintain correct replication. Depletion of WEE1 results in the formation of DSBs specifically in newly replicated DNA, as visualised in a modified pulse field electrophoresis assay. We were also able to detect formation of replication-associated secondary DSBs following treatment with ionizing radiation (IR). These DSBs were further demonstrated as major substrates for IR induced HR.

Using the DNA fibre technique we investigated the effect of DNA alkylating agents on replication. We found that DNA methylations pose direct physical blocks to progressing replication forks causing them to stall in a checkpoint independent manner. Furthermore, we studied restart kinetics following methylation blocked replication and identified a distinct restart mechanism for blocked replication forks independent of new origin firing and HR.

In conclusion, our findings increase the knowledge of replication dynamics following perturbed replication and further clarify the role of HR following IR induced damage and DNA alkylation.

Place, publisher, year, edition, pages
Stockholm: Department of Genetics, Microbiology and Toxicology, Stockholm University, 2011. 47 p.
DNA damage, DNA replication, homologous recombination, DNA double strand-breaks, ionizing radiation, alkylating agents
National Category
Natural Sciences Genetics
Research subject
Molecular Genetics
urn:nbn:se:su:diva-55048 (URN)978-91-7447-243-1 (ISBN)
Public defence
2011-03-31, lecture room G, Arrheniuslaboratorierna, Svante Arrhenius väg 20 C, Stockholm, 10:00 (English)
At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 4: Manuscript.Available from: 2011-03-09 Created: 2011-02-25 Last updated: 2011-02-28Bibliographically approved

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Groth, PetraHelleday, Thomas
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