A Cell Cycle-Specific Requirement for the XRCC1 BRCT II Domain during Mammalian DNA Strand Break Repair

Abstract

1 . This process correlates with the appearance of XRCC1 nuclear foci that colocalize with Rad51 and may thus function in concert with homologous recombination.DNA strand breakage can result in chromosomal rearrangement and is a major threat to genetic stability. Of particular threat are breaks that arise from damaged DNA bases, several thousand of which occur spontaneously per cell each day (20). The most common such breaks are single-strand breaks, which are formed as intermediates of base excision repair. The threat to genetic stability from DNA strand breaks that arise from base damage is illustrated by the phenotype of rodent cells that harbor mutations within the DNA repair gene XRCC1. XRCC1 is essential for embryonic development in mice (35), and XRCC1 mutant mouse or CHO cells that possess little or no XRCC1 protein exhibit increased frequencies of spontaneous sister chromatid exchange and chromosomal aberration (6,7,12,29,35,39). XRCC1 mutant cells appear unable to efficiently rejoin DNA single-strand breaks resulting from either endogenous base damage (35) or that induced by ionizing radiation or alkylating agents (36,37,39). Sequence analysis has not provided any indication of the role of XRCC1 in single-strand break repair (SSBR). However, biochemical studies have revealed that this protein interacts with DNA ligase III and DNA polymerase ␤ (6-8, 18). Thus, it is possible that XRCC1 functions as a molecular chaperone or scaffold protein, stabilizing and/or modifying the activity of other polypeptides. For example, the interaction of XRCC1 with DNA ligase III appears to be required for normal cellular levels of the latter, and reduced levels of DNA ligase III can result in inefficient SSBR during the excision repair of abasic sites in vitro (6,7,11). This interaction is mediated by a C-terminal BRCT domain in XRCC1, designated BRCT II (23, 34). BRCT domains have been identified in more than 40 polypeptides, defining a novel protein superfamily, and typically span 80 to 100 amino acids (5, 10). The function of these structures appears to involve, but may not be restricted to, the mediation of protein-protein interactions (5,10,23,34). In the present study, we have examined the importance of the XRCC1 BRCT II domain and its interaction with DNA ligase III to SSBR in Chinese hamster ovary (CHO) cells. MATERIALS AND METHODS Expression constructs, cell lines, and cell synchrony. A mutant XRCC1pmBRCT open reading frame (ORF) was generated by subcloning constructs described previously (34) and was inserted into the mammalian expression vector pcD2E (17). All XRCC1 ORFs encode a decahistidine tag at the C terminus. XRCC1 mutant EM9 cells were transfected with pcD2E or pcD2E harboring XRCC1 by electroporation, and Ͼ50 independent G418 r clones were pooled to generate the cell lines EM9-X, EM9-X pmBRCT , and EM9-V. Synchrony in G 1 was achieved via serum starvation and incubation in mimosine (25). Cells were incubated sequentially in ␣-MEM plus 10% fetal bovine serum (FBS) for 12 to 15 h, ␣-MEM plus 0.1% FBS fo...

Base excision repair of adenine/8-oxoguanine mispairs by an aphidicolin-sensitive DNA polymerase in human cell extracts

“…However, current data suggest that post-replicative BER-mediated removal of misincorporated bases (e.g. uracil) (Otterlei et al, 1999) or lesions that persist into S phase and might block replication (Taylor et al, 2000) occurs close to the replication fork.…”

Mutation of a BRCT domain selectively disrupts DNA single-strand break repair in noncycling Chinese hamster ovary cells

“…One of the proteins with which XRCC1 interacts is DNA Lig-3 (3,4). This interaction is mediated via BRCT domains located at the C terminus of both proteins (5, 6), and we have reported previously that the XRCC1 BRCT domain (denoted BRCT II) is important for SSBR during G 1 (7). However, this requirement is dispensable for cell survival in cycling cells because of an ability of XRCC1 to mediate SSBR independently of this domain in S͞G 2 (7).…”

“…This interaction is mediated via BRCT domains located at the C terminus of both proteins (5, 6), and we have reported previously that the XRCC1 BRCT domain (denoted BRCT II) is important for SSBR during G 1 (7). However, this requirement is dispensable for cell survival in cycling cells because of an ability of XRCC1 to mediate SSBR independently of this domain in S͞G 2 (7). Here, we have thus examined the possibility that the major role of the BRCT II domain is to mediate SSBR in noncycling cells, which lack an S͞G 2 cell-cycle phase and may thus be dependent on this domain for SSBR.…”

“…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, individually.…”

“…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?…”