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CK2 phosphorylation of XRCC1 facilitate single-strand break formation during base excision repair
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology. (Thomas Helleday)
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology. (Dag Jenssen)
(Grigory Dianov)
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology. (Thomas Helleday)
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Casein kinase 2 (CK2) phosphorylates the scaffold protein XRCC1, which is required for efficient DNA single-strand break (SSB) repair. Here, we express a XRCC1 protein (XRCC1ckm) that cannot be phosphorylated by CK2 in XRCC1 mutated EM9 cells and show that the role of this post-translational modification in SSB repair is distinct from its role in base excision repair (BER). Interestingly, we find that fewer SSBs are formed during BER after treatment with the alkylating agent dimethyl sulfate (DMS) in EM9 cells expressing XRCC1ckm (CKM cells) or following inhibition with CK2 inhibitor 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT). We also show that XRCC1ckm protein has a higher affinity for nicked DNA substrate than wild type XRCC1 protein and we propose a model whereby the increased affinity for DNA sequesters XRCC1ckm and the repair enzymes associated with it, at the repair site. In conclusion, our results indicate that CK2-phosphorylations of XRCC1 affect the kinetics of SSB repair and BER differentially, and that the modifications on XRCC1 facilitate the BER incision step.

Keyword [en]
CK2, XRCC1, dimethyl sulfate, base excision repair, single-strand break
National Category
Cell and Molecular Biology
Research subject
Molecular Genetics
Identifiers
URN: urn:nbn:se:su:diva-55790OAI: oai:DiVA.org:su-55790DiVA: diva2:406712
Available from: 2011-03-28 Created: 2011-03-28 Last updated: 2011-03-29Bibliographically 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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