Poly(ADP-ribose) polymerase-1 modulates DNA repair capacity and prevents formation of DNA double strand breaks.

Although poly(ADP-ribose) polymerase-1 (PARP-1) has no enzymatic activity involved in DNA damage processing by the base excision repair (BER) pathway, PARP-1 deficient cells are genetically unstable and sensitive to DNA-damaging agents. To explain this paradox, we investigated the impact of PARP-1 on BER in mammalian cells. We reduced cellular PARP-1 protein levels using siRNA, then introduced DNA damage by hydrogen peroxide treatment and examined the repair response. We find that PARP-1 is not involved in recruitment of the major BER proteins to sites of DNA damage. However, we find that PARP-1 protects excessive DNA single strand breaks (SSBs) from converting into DNA double strand breaks (DSBs) thus preserving them for subsequent repair by BER enzymes. This suggests that PARP-1 plays an important role in BER by extending the ability of BER enzymes to process DNA single strand breaks arising directly after mutagen stress or during processing of DNA lesions following extensive DNA damage

End-damage-specific proteins facilitate recruitment or stability of X-ray cross-complementing protein 1 at the sites of DNA single-strand break repair.

Ionizing radiation, oxidative stress and endogenous DNA-damage processing can result in a variety of single-strand breaks with modified 5' and/or 3' ends. These are thought to be one of the most persistent forms of DNA damage and may threaten cell survival. This study addresses the mechanism involved in recognition and processing of DNA strand breaks containing modified 3' ends. Using a DNA-protein cross-linking assay, we followed the proteins involved in the repair of oligonucleotide duplexes containing strand breaks with a phosphate or phosphoglycolate group at the 3' end. We found that, in human whole cell extracts, end-damage-specific proteins (apurinic/apyrimidinic endonuclease 1 and polynucleotide kinase in the case of 3' ends containing phosphoglycolate and phosphate, respectively) which recognize and process 3'-end-modified DNA strand breaks are required for efficient recruitment of X-ray cross-complementing protein 1-DNA ligase IIIalpha heterodimer to the sites of DNA repair

USP47 is a deubiquitylating enzyme that regulates base excision repair by controlling steady-state levels of DNA polymerase β.

DNA base excision repair (BER) is an essential cellular process required for genome stability, and misregulation of BER is linked to premature aging, increased rate of mutagenesis, and cancer. We have now identified the cytoplasmic ubiquitin-specific protease USP47 as the major enzyme involved in deubiquitylation of the key BER DNA polymerase (Pol β) and demonstrate that USP47 is required for stability of newly synthesized cytoplasmic Pol β that is used as a source for nuclear Pol β involved in DNA repair. We further show that knockdown of USP47 causes an increased level of ubiquitylated Pol β, decreased levels of Pol β, and a subsequent deficiency in BER, leading to accumulation of DNA strand breaks and decreased cell viability in response to DNA damage. Taken together, these data demonstrate an important role for USP47 in regulating DNA repair and maintaining genome integrity

Phosphorylation of PNKP by ATM prevents its proteasomal degradation and enhances resistance to oxidative stress.

We examined the mechanism regulating the cellular levels of PNKP, the major kinase/phosphatase involved in the repair of oxidative DNA damage, and find that it is controlled by ATM phosphorylation and ubiquitylation-dependent proteasomal degradation. We discovered that ATM-dependent phosphorylation of PNKP at serines 114 and 126 in response to oxidative DNA damage inhibits ubiquitylation-dependent proteasomal degradation of PNKP, and consequently increases PNKP stability that is required for DNA repair. We have also purified a novel Cul4A-DDB1 ubiquitin ligase complex responsible for PNKP ubiquitylation and identify serine-threonine kinase receptor associated protein (STRAP) as the adaptor protein that provides specificity of the complex to PNKP. Strap(-/-) mouse embryonic fibroblasts subsequently contain elevated cellular levels of PNKP, and show elevated resistance to oxidative DNA damage. These data demonstrate an important role for ATM and the Cul4A-DDB1-STRAP ubiquitin ligase in the regulation of the cellular levels of PNKP, and consequently in the repair of oxidative DNA damage

XRCC1 phosphorylation by CK2 is required for its stability and efficient DNA repair.

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 IIIalpha (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

Ubiquitin ligase ARF-BP1/Mule modulates base excision repair

Base excision repair (BER) is the major cellular pathway involved in removal of endogenous/spontaneous DNA lesions. Here, we study the mechanism that controls the steady-state levels of BER enzymes in human cells. By fractionating human cell extract, we purified the E3 ubiquitin ligase Mule (ARF-BP1/HectH9) as an enzyme that can ubiquitylate DNA polymerase β (Pol β), the major BER DNA polymerase. We identified lysines 41, 61 and 81 as the major sites of modification and show that replacement of these lysines to arginines leads to increased protein stability. We further show that the cellular levels of Pol β and its ubiquitylated derivative are modulated by Mule and ARF and siRNA knockdown of Mule leads to accumulation of Pol β and increased DNA repair. Our findings provide a novel mechanism regulating steady-state levels of BER proteins