ARF induction in response to DNA strand breaks is regulated by PARP1.

The ARF tumour suppressor protein, the gene of which is frequently mutated in many human cancers, plays an important role in the cellular stress response by orchestrating up-regulation of p53 protein and consequently promoting cell-cycle delay. Although p53 protein function has been clearly linked to the cellular DNA damage response, the role of ARF protein in this process is unclear. Here, we report that arf gene transcription is induced by DNA strand breaks (SBs) and that ARF protein accumulates in response to persistent DNA damage. We discovered that poly(ADP-ribose) synthesis catalysed by PARP1 at the sites of unrepaired SBs activates ARF transcription through a protein signalling cascade, including the NAD(+)-dependent deacetylase SIRT1 and the transcription factor E2F1. Our data suggest that poly(ADP-ribose) synthesis at the sites of SBs initiates DNA damage signal transduction by reducing the cellular concentration of NAD(+), thus down-regulating SIRT1 activity and consequently activating E2F1-dependent ARF transcription. Our findings suggest a vital role for ARF in DNA damage signalling, and furthermore explain the critical requirement for ARF inactivation in cancer cells, which are frequently deficient in DNA repair and accumulate DNA damage.

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.

ATM-dependent downregulation of USP7/HAUSP by PPM1G activates p53 response to DNA damage.

The deubiquitylation enzyme USP7/HAUSP plays a major role in regulating genome stability and cancer prevention by controlling the key proteins involved in the DNA damage response. Despite this important role in controlling other proteins, USP7 itself has not been recognized as a target for regulation. Here, we report that USP7 regulation plays a central role in DNA damage signal transmission. We find that stabilization of Mdm2, and correspondingly p53 downregulation in unstressed cells, is accomplished by a specific isoform of USP7 (USP7S), which is phosphorylated at serine 18 by the protein kinase CK2. Phosphorylation stabilizes USP7S and thus contributes to Mdm2 stabilization and downregulation of p53. After ionizing radiation, dephosphorylation of USP7S by the ATM-dependent protein phosphatase PPM1G leads to USP7S downregulation, followed by Mdm2 downregulation and accumulation of p53. Our findings provide a quantitative transmission mechanism of the DNA damage signal to coordinate a p53-dependent DNA damage response.

Ubiquitin ligase UBR3 regulates cellular levels of the essential DNA repair protein APE1 and is required for genome stability.

APE1 (Ref-1) is an essential human protein involved in DNA damage repair and regulation of transcription. Although the cellular functions and biochemical properties of APE1 are well characterized, the mechanism involved in regulation of the cellular levels of this important DNA repair/transcriptional regulation enzyme, remains poorly understood. Using an in vitro ubiquitylation assay, we have now purified the human E3 ubiquitin ligase UBR3 as a major activity that polyubiquitylates APE1 at multiple lysine residues clustered on the N-terminal tail. We further show that a knockout of the Ubr3 gene in mouse embryonic fibroblasts leads to an up-regulation of the cellular levels of APE1 protein and subsequent genomic instability. These data propose an important role for UBR3 in the control of the steady state levels of APE1 and consequently error free DNA repair.

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

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 ß) and demonstrate that USP47 is required for stability of newly synthesized cytoplasmic Pol ß that is used as a source for nuclear Pol ß involved in DNA repair. We further show that knockdown of USP47 causes an increased level of ubiquitylated Pol ß, decreased levels of Pol ß, 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.

USP7/HAUSP stimulates repair of oxidative DNA lesions.

USP7 is involved in the cellular stress response by regulating Mdm2 and p53 protein levels following severe DNA damage. In addition to this, USP7 may also play a role in chromatin remodelling by direct deubiquitylation of histones, as well as indirectly by regulating the cellular levels of E3 ubiquitin ligases involved in histone ubiquitylation. Here, we provide new evidence that USP7 modulated chromatin remodelling is important for base excision repair of oxidative lesions. We show that transient USP7 siRNA knockdown did not change the levels or activity of base excision repair enzymes, but significantly reduced chromatin DNA accessibility and consequently the rate of repair of oxidative lesions.

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 beta (Pol beta), 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 beta and its ubiquitylated derivative are modulated by Mule and ARF and siRNA knockdown of Mule leads to accumulation of Pol beta and increased DNA repair. Our findings provide a novel mechanism regulating steady-state levels of BER proteins.

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.

CHIP-mediated degradation and DNA damage-dependent stabilization regulate base excision repair proteins.

Base excision repair (BER) is the major pathway for processing of simple lesions in DNA, including single-strand breaks, base damage, and base loss. The scaffold protein XRCC1, DNA polymerase beta, and DNA ligase IIIalpha play pivotal roles in BER. Although all these enzymes are essential for development, their cellular levels must be tightly regulated because increased amounts of BER enzymes lead to elevated mutagenesis and genetic instability and are frequently found in cancer cells. Here we report that BER enzyme levels are linked to and controlled by the level of DNA lesions. We demonstrate that stability of BER enzymes increases after formation of a repair complex on damaged DNA and that proteins not involved in a repair complex are ubiquitylated by the E3 ubiquitin ligase CHIP and subsequently rapidly degraded. These data identify a molecular mechanism controlling cellular levels of BER enzymes and correspondingly the efficiency and capacity of BER.

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.

DNA polymerase beta promotes recruitment of DNA ligase III alpha-XRCC1 to sites of base excision repair.

Base excision repair is a major pathway for the removal of simple lesions in DNA including base damage and base loss (abasic site). Base excision repair requires the coordinated action of several repair and ancillary proteins, the impairment of which can lead to genetic instability. Using a protein-DNA cross-linking assay during repair in human whole cell extracts, we monitored proteins involved in the initial steps of repair of a substrate containing a site-specific abasic site to address the molecular events following incision of the abasic site by AP endonuclease. We find that after dissociation of AP endonuclease from the incised abasic site, both DNA polymerase beta (Pol beta) and the DNA ligase IIIalpha-XRCC1 heterodimer efficiently bind/cross-link to the substrate DNA. We also find that the cross-linking efficacy of the DNA ligase IIIalpha-XRCC1 heterodimer was decreased about 2-fold in the Pol beta-deficient cell extract but was rescued by addition of purified wild type but not a mutant Pol beta protein that does not interact with the DNA ligase IIIalpha-XRCC1 heterodimer. We further demonstrate that Pol beta and the DNA ligase IIIalpha-XRCC1 heterodimer are present at equimolar concentrations in whole cell extracts and that Pol beta has a 7-fold higher affinity to the incised abasic site containing substrate than DNA ligase IIIalpha. Using gel filtration of whole cell extracts prepared at physiological salt conditions (0.15 M NaCl), we find no evidence for a stable preexisting complex of DNA Pol beta with the DNA ligase IIIalpha-XRCC1 heterodimer. Taken together, these data suggest that following incision by AP endonuclease, DNA Pol beta recognizes and binds to the incised abasic site and promotes recruitment of the DNA ligase IIIalpha-XRCC1 heterodimer through its interaction with XRCC1.

Poly(ADP-ribose) polymerase-1 protects excessive DNA strand breaks from deterioration during repair in human cell extracts.

Base excision repair (BER), a major pathway for the removal of simple lesions in DNA, requires the co-ordinated action of several repair and ancillary proteins, the impairment of which can lead to genetic instability. We here address the role of poly(ADP-ribose) polymerase-1 (PARP-1) in BER. Using an in vitro cross-linking assay, we reveal that PARP-1 is always involved in repair of a uracil-containing oligonucleotide and that it binds to the damaged DNA during the early stages of repair. Inhibition of PARP-1 poly(ADP-ribosyl)ation by 3-aminobenzamide blocks dissociation of PARP-1 from damaged DNA and prevents further repair. We find that excessive poly(ADP-ribosyl)ation occurs when repair intermediates containing single-strand breaks are in excess of the repair capacity of the cell extract, suggesting that repeated binding of PARP-1 to the nicked DNA occurs. We also find increased sensitivity of repair intermediates to nuclease cleavage in PARP-deficient mouse fibroblasts and after depletion of PARP-1 from HeLa whole cell extracts. Our data support the model in which PARP-1 binding to DNA single-strand breaks or repair intermediates plays a protective role when repair is limited.

APE1-dependent repair of DNA single-strand breaks containing 3'-end 8-oxoguanine.

DNA single-strand breaks containing 3'-8-oxoguanine (3'-8-oxoG) ends can arise as a consequence of ionizing radiation and as a result of DNA polymerase infidelity by misincorporation of 8-oxodGMP. In this study we examined the mechanism of repair of 3'-8-oxoG within a single-strand break using purified base excision repair enzymes and human whole cell extracts. We find that 3'-8-oxoG inhibits ligation by DNA ligase IIIalpha or DNA ligase I, inhibits extension by DNA polymerase beta and that the lesion is resistant to excision by DNA glycosylases involved in the repair of oxidative lesions in human cells. However, we find that purified human AP-endonuclease 1 (APE1) is able to remove 3'-8-oxoG lesions. By fractionation of human whole cell extracts and immunoprecipitation of fractions containing 3'-8-oxoG excision activity, we further demonstrate that APE1 is the major activity involved in the repair of 3'-8-oxoG lesions in human cells and finally we reconstituted repair of the 3'-8-oxoG-containing oligonucleotide duplex with purified human enzymes including APE1, DNA polymerase beta and DNA ligase IIIalpha.

APE1 is the major 3'-phosphoglycolate activity in human cell extracts.

DNA strand breaks containing 3'-phosphoglycolate (3'-PG) ends are the major lesions induced by ionizing radiation. The repair of this lesion is not completely understood and several activities are thought to be involved in processing of 3'-PG ends. In this study we examined activities in human whole cell extracts (WCE) responsible for removal of 3'-PG. Using a radiolabelled oligonucleotide containing a single nucleotide gap with internal 5'-phosphate and 3'-PG ends, we demonstrate that the major 3'-PG activity in human WCE is Mg2+ dependent and that this activity co-purifies with AP endonuclease 1 (APE1) over phosphocellulose and gel filtration chromatography. Furthermore, immunodepletion of APE1 from active gel filtration fractions using APE1 specific antibodies reveals that the major activity against 3'-PG in human WCE is APE1.

XRCC1-DNA polymerase beta interaction is required for efficient base excision repair.

X-ray repair cross-complementing protein-1 (XRCC1)-deficient cells are sensitive to DNA damaging agents and have delayed processing of DNA base lesions. In support of its role in base excision repair, it was found that XRCC1 forms a tight complex with DNA ligase IIIalpha and also interacts with DNA polymerase beta (Pol beta) and other base excision repair (BER) proteins. We have isolated wild-type XRCC1-DNA ligase IIIalpha heterodimer and mutated XRCC1-DNA ligase IIIalpha complex that does not interact with Pol beta and tested their activities in BER reconstituted with human purified proteins. We find that a point mutation in the XRCC1 protein which disrupts functional interaction with Pol beta, affected the ligation efficiency of the mutant XRCC1-DNA ligase IIIalpha heterodimer in reconstituted BER reactions. We also compared sensitivity to hydrogen peroxide between wild-type CHO-9 cells, XRCC1-deficient EM-C11 cells and EM-C11 cells transfected with empty plasmid vector or with plasmid vector carrying wild-type or mutant XRCC1 gene and find that the plasmid encoding XRCC1 protein, that does not interact with Pol beta has reduced ability to rescue the hydrogen peroxide sensitivity of XRCC1- deficient cells. These data suggest an important role for the XRCC1-Pol beta interaction for coordinating the efficiency of the BER process.

Repair of abasic sites in DNA.

Repair of both normal and reduced AP sites is activated by AP endonuclease, which recognizes and cleaves a phosphodiester bond 5' to the AP site. For a short period of time an incised AP site is occupied by poly(ADP-ribose) polymerase and then DNA polymerase beta adds one nucleotide into the repair gap and simultaneously removes the 5'-sugar phosphate. Finally, the DNA ligase III/XRCC1 complex accomplishes repair by sealing disrupted DNA ends. However, long-patch BER pathway, which is involved in the removal of reduced abasic sites, requires further DNA synthesis resulting in strand displacement and the generation of a damage-containing flap that is later removed by the flap endonuclease. Strand-displacement DNA synthesis is accomplished by DNA polymerase delta/epsilon and DNA ligase I restores DNA integrity. DNA synthesis by DNA polymerase delta/epsilon is dependent on proliferating cell nuclear antigen, which also stimulates the DNA ligase I and flap endonuclease. These repair events are supported by multiple protein-protein interactions.

Poly(ADP-ribose) polymerase in base excision repair: always engaged, but not essential for DNA damage processing.

Poly(ADP-ribose) polymerase (PARP-1) is an abundant nuclear protein with a high affinity for single- and double-strand DNA breaks. Its binding to strand breaks promotes catalysis of the covalent modification of nuclear proteins with poly(ADP-ribose) synthesised from NAD(+). PARP-1-knockout cells are extremely sensitive to alkylating agents, suggesting the involvement of PARP-1 in base excision repair; however, its role remains unclear. We investigated the dependence of base excision repair pathways on PARP-1 and NAD(+) using whole cell extracts derived from normal and PARP-1 deficient mouse cells and DNA substrates containing abasic sites. In normal extracts the rate of repair was highly dependent on NAD(+). We found that in the absence of NAD(+) repair was slowed down 4-6-fold after incision of the abasic site. We also established that in extracts from PARP-1 deficient mouse cells, repair of both regular and reduced abasic sites was increased with respect to normal extracts and was NAD(+)-independent, suggesting that in both short- and long-patch BER PARP-1 slows down, rather than stimulates, the repair reaction. Our data support the proposal that PARP-1 does not play a major role in catalysis of DNA damage processing via either base excision repair pathway.

Repair of clustered DNA lesions. Sequence-specific inhibition of long-patch base excision repair be 8-oxoguanine.

Ionizing radiation induces clustered DNA damage where two or more lesions are located proximal to each other on the same or opposite DNA strands. It has been suggested that individual lesions within a cluster are removed sequentially and that the presence of a vicinal lesion(s) may affect the rate and fidelity of DNA repair. In this study, we addressed the question of how 8-oxoguanine located opposite to normal or reduced abasic sites would affect the repair of these sites by the base excision repair system. We have found that an 8-oxoguanine located opposite to an abasic site does not affect either the efficiency or fidelity of repair synthesis by DNA polymerase beta. In contrast, an 8-oxoguanine located one nucleotide 3'-downstream of the abasic site significantly reduces both strand displacement synthesis supported by DNA polymerase beta or delta and cleavage by flap endonuclease of the generated flap, thus inhibiting the long-patch base excision repair pathway.