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XRCC1 phosphorylation by CK2 is required for its stability and efficient DNA repair
University of Oxford, Gray Institute for Radiation Oncology & Biology.
University of Oxford, Gray Institute for Radiation Oncology & Biology.
University of Oxford, Gray Institute for Radiation Oncology & Biology.
University of Oxford, Gray Institute for Radiation Oncology & Biology.
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2010 (English)In: DNA Repair, ISSN 1568-7864, E-ISSN 1568-7856, Vol. 9, no 7, 835-841 p.Article in journal (Refereed) Published
Abstract [en]

XRCC1 is a scaffold protein that interacts with several DNA repair proteins and plays a critical role in DNA base excision repair (BER). XRCC1 protein is in a tight complex with DNA ligase III alpha (Lig III) and this complex is involved in the ligation step of both BER and repair of DNA single strand breaks. The majority of XRCC1 has previously been demonstrated to exist in a phosphorylated form and cells containing mutant XRCC1, that is unable to be phosphorylated, display a reduced rate of single strand break repair. Here, in an unbiased assay, we demonstrate that the cytoplasmic form of the casein kinase 2 (CK2) protein is the major protein kinase activity involved in phosphorylation of XRCC1 in human cell extracts and that XRCC1 phosphorylation is required for XRCC1-Lig III complex stability. We demonstrate that XRCC1-Lig III complex containing mutant XRCC1, in which CK2 phosphorylation sites have been mutated, is unstable. We also find that a knockdown of CK2 by siRNA results in both reduced XRCC1 phosphorylation and stability, which also leads to a reduced amount of Lig III and accumulation of DNA strand breaks. We therefore propose that CK2 plays an important role in DNA repair by contributing to the stability of XRCC1-Lig III complex.

Place, publisher, year, edition, pages
2010. Vol. 9, no 7, 835-841 p.
Keyword [en]
Base excision repair, XRCC1, Phosphorylation, Protein stability
National Category
Genetics
Research subject
Genetics
Identifiers
URN: urn:nbn:se:su:diva-50893DOI: 10.1016/j.dnarep.2010.04.008ISI: 000279964200012OAI: oai:DiVA.org:su-50893DiVA: diva2:383364
Note
authorCount :7Available from: 2011-01-04 Created: 2011-01-03 Last updated: 2017-12-11Bibliographically approved
In thesis
1. Post-translational modifications in DNA base excision repair: The roles of CK2 and PARP-1
Open this publication in new window or tab >>Post-translational modifications in DNA base excision repair: The roles of CK2 and PARP-1
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Base lesions and DNA single-strand breaks (SSBs) are very common types of DNA damage. The base excision repair (BER) and single-strand break repair (SSBR) machineries both require a succession of enzymatic events in order to remove these types of endogenous lesions and to restore the DNA. Coordinated repair involves signalling between the proteins concerned and is achieved by post-translational modification. Here, we study two types of modifications in the context of BER and SSBR.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a known SSB sensor, which utilizes NAD+ and converts these to ADP-ribose polymers as a post-translational modification of primarily itself, to accelerate repair. However, its role in BER is not as clear. By quantification of SSBs in vivo, we find that PARP inhibition prevents the completion of BER, while siRNA knockdown of PARP-1 leaves repair unaffected. Our results indicate that PARP-1 is not required for BER to progress, but that the enzyme interferes with the SSB intermediate.

Another known post-translational modification in SSBR is the phosphorylation of XRCC1 by CK2. Here, we show that the majority of the cellular XRCC1 is phosphorylated and that CK2 is the main kinase responsible for this. We find that this modification prevents degradation of XRCC1 by the proteasome, resulting in faster repair of oxidative damage in the DNA. In addition, we propose a new role for CK2 modifications of XRCC1 in BER. We demonstrate that, even though the presence of XRCC1 or the activity of PARP are not required for SSB intermediate formation, the expression of a non-phosphorylated form of XRCC1 results in reduced SSB levels. Furthermore, the affinity of XRCC1 for a nicked DNA substrate increases when the CK2 phosphorylation sites are mutated.

To summarise, our findings increase the knowledge of the BER and SSBR processes and demonstrate that the impact of post-translational modifications is more complex than it originally appeared.

Place, publisher, year, edition, pages
Stockholm: Department of Genetics, Microbiology and Toxicology, Stockholm University, 2011. 55 p.
Keyword
base excision repair, single-strand break repair, PARP-1, CK2, XRCC1, mammalian cells
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:su:diva-55792 (URN)978-91-7447-267-7 (ISBN)
Public defence
2011-04-29, sal G, Arrheniuslaboratorierna, Svante Arrhenius väg 20 C, Stockholm, 10:00 (English)
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Note
At the time of doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.Available from: 2011-04-07 Created: 2011-03-28 Last updated: 2011-04-04Bibliographically approved

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