DNA 3′-Phosphatase Activity Is Critical for Rapid Global Rates of Single-Strand Break Repair following Oxidative Stress

Abstract

Oxidative stress is a major source of chromosome single-strand breaks (SSBs), and the repair of these lesions is retarded in neurodegenerative disease. The rate of the repair of oxidative SSBs is accelerated by XRCC1, a scaffold protein that is essential for embryonic viability and that interacts with multiple DNA repair proteins. However, the relative importance of the interactions mediated by XRCC1 during oxidative stress in vivo is unknown. We show that mutations that disrupt the XRCC1 interaction with DNA polymerase ␤ or DNA ligase III fail to slow SSB repair in proliferating CHO cells following oxidative stress. In contrast, mutation of the domain that interacts with polynucleotide kinase/phosphatase (PNK) and Aprataxin retards repair, and truncated XRCC1 encoding this domain fully supports this process. Importantly, the impact of mutating the protein domain in XRCC1 that binds these end-processing factors is circumvented by the overexpression of wild-type PNK but not by the overexpression of PNK harboring a mutated DNA 3-phosphatase domain. These data suggest that DNA 3-phosphatase activity is critical for rapid rates of chromosomal SSB repair following oxidative stress, and that the XRCC1-PNK interaction ensures that this activity is not rate limiting in vivo.Oxidative stress can have a major influence on genome integrity and cell survival and is an etiological factor in a number of neurological human diseases. Of these, several are associated with defects in the repair of DNA damage, including xeroderma pigmentosum (XP), ataxia telangiecatsia (A-T), ataxia oculomotor apraxia 1 (AOA1), and spinocerebellar ataxia with axonal neuropathy 1 (SCAN1) (1,28,34,37). The neuropathology evident in XP most likely reflects an inability to repair one or more single-strand oxidative adducts by nucleotide excision repair. In contrast, A-T is associated with cellular defects in the repair of DNA double-strand breaks (DSBs) and AOA1 and SCAN1 with defects in the repair of DNA single-strand breaks (SSBs). SSBs are the commonest DNA lesions arising in cells, and if they are not rapidly repaired they can inhibit transcription and/or generate replication-associated DSBs (3,26,59,60). The repair of oxidative SSBs involves DNA damage detection by PARP-1 followed by recruitment of the enzymes required for subsequent steps of the repair process, which include DNA end processing, DNA gap filling, and DNA ligation (9, 19). Many of the enzymes implicated in these steps interact physically with XRCC1, including DNA polynucleotide kinase (PNK) (54), Aprataxin (APTX) (13,14,18,31,44), DNA polymerase ␤ (Pol ␤) (10, 27), and DNA ligase III␣ (Lig3␣) (11,12). This has prompted the hypothesis that XRCC1 is a scaffold protein that recruits, stabilizes, and/or stimulates SSB repair (SSBR) enzymes at chromosomal SSBs, thereby accelerating the overall process (8, 9). While in vitro analyses generally are consistent with this idea, including the observation that XRCC1 mutation (50, 58), deletion (49), or depletion (6) retards the rate of chromos...

Disconnecting XRCC1 and DNA ligase III

“…Consistent with our data, normal repair is observed after oxidative damage when the XRCC1-LIG3 interaction is disabled 63 and knockdown of LIG3 via RNA interference results in repair deficiency after UV damage. 64 Furthermore, a companion study to our own used nuclear Lig3-deficient ES cells complemented with a mitochondrial version of Lig3 to maintain cellular viability and showed survival of these ES cells was similar to controls after an assortment of genotoxic agents.…”

DNA strand break repair and neurodegeneration

“…PARP1 is the sensor of chromosomal SSBs, and results in accumulation of SSBR proteins such as the XRCC1/DNA Ligase III (Lig3) heterodimer at sites of DNA damage [107,108]. XRCC1/Lig3 appears to assemble or recruit a number of DNA processing factors at sites of SSBs, such as Aprataxin, TDP1, PNKP, and DNA pol , [109][110][111]. In mice, Lig3 is essential for mitochondrial DNA maintenance and embryonic viability [112,113].…”

“…In this case the DNA substrates are 5'-hydroxyl and/or 3'-phosphate termini, at either SSBs [110,133] or DSBs [132,134], which require the DNA kinase or DNA phosphatase activity of PNKP to restore ligatable 5'-phosphate and 3'-hydroxyl termini, respectively [135]. In a similar manner to aprataxin and TDP1, loss of PNKP phosphatase activity results in delayed repair of cellular SSBs, and sensitivity to oxidative stress [109,136]. Patients with mutations in this protein suffer from microcephaly, early-onset seizures and developmental delay (MCSZ [137]).…”

The Interaction between Polynucleotide Kinase Phosphatase and the DNA Repair Protein XRCC1 Is Critical for Repair of DNA Alkylation Damage and Stable Association at DNA Damage Sites

“…Proteins-In initial studies, we examined the effect of DNA ligase III␣-XRCC1 on PNKP 3Ј-phosphatase activity, which is required for the repair of single-strand breaks generated by oxidative DNA damage (38). The complex containing wild-type XRCC1 enhanced removal of the 3Ј-phosphate group by more than 6-fold at a 2:1 ratio of DNA ligase III␣-XRCC1 complex to PNKP, whereas the complex containing the A482T mutant did not significantly increase the 3Ј-phosphatase activity of PNKP under similar conditions (Fig.…”