Ataxia oculomotor apraxia-1 (AOA1) is a neurological disorder caused by mutations in the gene (APTX) encoding aprataxin. Aprataxin is a member of the histidine triad (HIT) family of nucleotide hydrolases and transferases, and inactivating mutations are largely confined to this HIT domain. Aprataxin associates with the DNA repair proteins XRCC1 and XRCC4, which are partners of DNA ligase III and ligase IV, respectively, suggestive of a role in DNA repair. Consistent with this, APTX-defective cell lines are sensitive to agents that cause single-strand breaks and exhibit an increased incidence of induced chromosomal aberrations. It is not, however, known whether aprataxin has a direct or indirect role in DNA repair, or what the physiological substrate of aprataxin might be. Here we show, using purified aprataxin protein and extracts derived from either APTX-defective chicken DT40 cells or Aptx-/- mouse primary neural cells, that aprataxin resolves abortive DNA ligation intermediates. Specifically, aprataxin catalyses the nucleophilic release of adenylate groups covalently linked to 5'-phosphate termini at single-strand nicks and gaps, resulting in the production of 5'-phosphate termini that can be efficiently rejoined. These data indicate that neurological disorders associated with APTX mutations may be caused by the gradual accumulation of unrepaired DNA strand breaks resulting from abortive DNA ligation events.

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The mechanisms by which the progression of eukaryotic replication forks is controlled after DNA damage are unclear. We have found that fork progression is slowed by cisplatin or UV treatment in intact vertebrate cells and in replication assays in vitro. Fork slowing is reduced or absent in irs1SF CHO cells and XRCC3(-/-) chicken DT40 cells, indicating that fork slowing is an active process that requires the homologous recombination protein XRCC3. The addition of purified human Rad51C-XRCC3 complex restores fork slowing in permeabilized XRCC3(-/-) cells. Moreover, the requirement for XRCC3 for fork slowing can be circumvented by addition of human Rad51. These data demonstrate that the recombination proteins XRCC3 and Rad51 cooperatively modulate the progression of replication forks on damaged vertebrate chromosomes.

Ataxia oculomotor apraxia 1 (AOA1) results from mutations in aprataxin, a component of DNA strand break repair that removes AMP from 5 termini. Despite this, global rates of chromosomal strand break repair are normal in a variety of AOA1 and other aprataxin-defective cells. Here we show that short-patch single-strand break repair (SSBR) in AOA1 cell extracts bypasses the point of aprataxin action at oxidative breaks and stalls at the final step of DNA ligation, resulting in the accumulation of adenylated DNA nicks. Strikingly, this defect results from insufficient levels of nonadenylated DNA ligase, and short-patch SSBR can be restored in AOA1 extracts, independently of aprataxin, by the addition of recombinant DNA ligase. Since adenylated nicks are substrates for long-patch SSBR, we reasoned that this pathway might in part explain the apparent absence of a chromosomal SSBR defect in aprataxin-defective cells. Indeed, whereas chemical inhibition of long-patch repair did not affect SSBR rates in wild-type mouse neural astrocytes, it uncovered a significant defect in Aptx ؊/؊ neural astrocytes. These data demonstrate that aprataxin participates in chromosomal SSBR in vivo and suggest that short-patch SSBR arrests in AOA1 because of insufficient nonadenylated DNA ligase.Oxidative stress is an etiological factor in many neurological diseases, including Alzheimer's disease, Parkinson's disease, and Huntington's disease. One type of macromolecule damaged by reactive oxygen species is DNA, and oxidative damage to DNA has been suggested to be a significant factor in these and other neurological conditions (2). In particular, a number of rare hereditary neurodegenerative disorders have provided direct support for the notion that unrepaired DNA damage causes neural dysfunction. Not least of these are the recessive spinocerebellar ataxias, a number of which are associated with mutations in DNA damage response proteins (17). The archetypal DNA damage-associated spinocerebellar ataxia is ataxiatelangiectasia (A-T), in which mutations in ATM protein result in defects in the detection and signaling of DNA double-strand breaks (DSBs) (3). A-T-like disorder is a related disease that exhibits neurological features similar to those of A-T, resulting from mutation of Mre11, a component of the MRN complex that operates in conjunction with ATM during DSB detection and signaling (28).Two additional spinocerebellar ataxias are spinocerebellar ataxia with axonal neuropathy 1 (SCAN1) and ataxia oculomotor apraxia 1 (AOA1), in which the TDP1 and aprataxin proteins are mutated, respectively (9,19,27). Both TDP1 and aprataxin are components of the DNA strand break repair machinery (recently reviewed in references 6 and 24). Whereas SCAN1 is currently limited to nine individuals from a single family, AOA1 is one of the commonest recessive spinocerebellar ataxias. Aprataxin is a member of the histidine triad superfamily of nucleotide hydrolases/transferases and has been reported to remove phosphate and phosphoglycolate moieties from the 3Ј te...

The DNA single-strand break repair protein XRCC1 contains a BRCT domain that binds and stabilizes intracellular DNA ligase III protein.We recently demonstrated that this domain is largely dispensable for single-strand break repair and cellular resistance to DNA base damage in cycling cells. Here, we report that the BRCT domain is required for single-strand break repair in noncycling cells. Mutations that disrupt the BRCT domain and prevent DNA ligase III interaction abolished XRCC1-dependent repair in serum-starved Chinese hamster ovary cells, and reentry into cell cycle induced by readdition of serum restored repair. Elevating DNA ligase III levels in XRCC1 mutant cells using proteosome inhibitors or by expressing XRCC1 protein in which the BRCT domain is disrupted but can still bind DNA ligase III failed to restore repair in noncycling cells. The requirement for the BRCT domain for DNA strand break repair is thus for more than simply binding and stabilizing DNA ligase III. These data provide evidence in support of a selective role for a DNA repair protein or protein domain in noncycling cells. We propose that the XRCC1 C-terminal BRCT domain may be important for genetic stability in postmitotic cells in vivo.T housands of DNA single-strand breaks arise in cells every day from a variety of sources including endogenous reactive oxygen species and the enzymatic excision of damaged DNA bases or abasic sites (1, 2). The threat posed by single-strand breaks is illustrated by the genetic instability of mutant rodent cells in which single-strand break repair (SSBR) is defective. For example, mutations in the SSBR gene XRCC1 result in increased frequencies of spontaneous sister chromatid exchange and chromosomal aberration as well as hypersensitivity to alkylating agents and ionizing radiation (see ref. 1 for review). XRCC1 protein interacts with DNA ligase III (Lig-3) and maintains normal cellular levels of this polypeptide (3, 4). The interaction between XRCC1 and Lig-3 is mediated via BRCT domains located at the C terminus of both polypeptides (5, 6). Intriguingly, there is a cell-cycle stage-specific requirement in mammalian cells for the C-terminal XRCC1 BRCT domain (denoted BRCT II), as it is required for XRCC1-dependent SSBR during G 1 but is dispensable for this process in S phase (7). However, the role in G 1 is largely dispensable for survival in cycling cells because XRCC1-dependent SSBR in S phase can largely compensate for an absence of repair in G 1 (7). If the BRCT II domain is dispensable in cycling cells, what role does it play? Here, we have examined the possibility that the major role for this motif is during SSBR in noncycling cells, which lack S phase and may thus be dependent on this BRCT domain for SSBR. Materials and MethodsExpression Constructs and Cell Lines. EM9-V cells harbor empty vector and EM9-X pmBRCT cells express human XRCC1 possessing the BRCT II domain mutations W611D and VI584͞585DD (7). EM9-X W611D and EM9-X VI584/585DD cells express human XRCC1 possessing either of these mutations...