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Post-translational modifications in DNA base excision repair: The roles of CK2 and PARP-1
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology. (Thomas Helleday)
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 [en]
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: urn:nbn:se:su:diva-55792ISBN: 978-91-7447-267-7 (print)OAI: oai:DiVA.org:su-55792DiVA: diva2:406721
Public defence
2011-04-29, sal G, Arrheniuslaboratorierna, Svante Arrhenius väg 20 C, Stockholm, 10:00 (English)
Opponent
Supervisors
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
List of papers
1. Poly (ADP-ribose) polymerase (PARP) is not involved in base excision repair but PARP inhibition traps a single-strand intermediate
Open this publication in new window or tab >>Poly (ADP-ribose) polymerase (PARP) is not involved in base excision repair but PARP inhibition traps a single-strand intermediate
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2011 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 39, no 8, 3166-3175 p.Article in journal (Refereed) Published
Abstract [en]

Base excision repair (BER) represents the most important repair pathway of endogenous DNA lesions. Initially, a base damage is recognized, excised and a DNA single-strand break (SSB) intermediate forms. The SSB is then ligated, a process that employs proteins also involved in SSB repair, e.g. XRCC1, Ligase III and possibly PARP1. Here, we confirm the role of XRCC1 and PARP in direct SSB repair. Interestingly, we uncover a synthetic lethality between XRCC1 deficiency and PARP inhibition. We also treated cells with alkylating agent dimethyl sulfate (DMS) and monitored the SSB intermediates formed during BER. DMS-induced SSBs were quickly repaired in wild-type cells; while a rapid accumulation of SSBs was observed in cells where post-incision repair was blocked by a PARP inhibitor or by XRCC1 deficiency (EM9 cells). Interestingly, DMS-induced SSBs did not accumulate in PARP1 siRNA depleted cells, demonstrating that PARP1 is not required for efficient completion of BER. Based on these results we suggest no immediate role for PARP1 in BER, but that PARP inhibitors trap PARP on the SSB intermediate formed during BER. Unexpectedly, addition of PARP inhibitor 2 h after DMS treatment still increased SSB levels indicating ongoing repair even at this late time point.

National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-55787 (URN)10.1093/nar/gkq1241 (DOI)000290055200021 ()21183466 (PubMedID)
Note
authorCount :6Available from: 2011-03-28 Created: 2011-03-28 Last updated: 2017-12-11Bibliographically approved
2. XRCC1 phosphorylation by CK2 is required for its stability and efficient DNA repair
Open this publication in new window or tab >>XRCC1 phosphorylation by CK2 is required for its stability and efficient DNA repair
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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.

Keyword
Base excision repair, XRCC1, Phosphorylation, Protein stability
National Category
Genetics
Research subject
Genetics
Identifiers
urn:nbn:se:su:diva-50893 (URN)10.1016/j.dnarep.2010.04.008 (DOI)000279964200012 ()
Note
authorCount :7Available from: 2011-01-04 Created: 2011-01-03 Last updated: 2017-12-11Bibliographically approved
3. CK2 phosphorylation of XRCC1 facilitate single-strand break formation during base excision repair
Open this publication in new window or tab >>CK2 phosphorylation of XRCC1 facilitate single-strand break formation during base excision repair
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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
CK2, XRCC1, dimethyl sulfate, base excision repair, single-strand break
National Category
Cell and Molecular Biology
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:su:diva-55790 (URN)
Available from: 2011-03-28 Created: 2011-03-28 Last updated: 2011-03-29Bibliographically approved

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