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

Abstract

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...

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

“…1B). We have shown previously that the deletion of the C-terminal BRCT domain ablates binding to Lig3␣ (36). Consistent with this, Lig3 levels were no higher in EM9-XH ␦529-633 cells than in EM9-V cells, consistent with the requirement for interaction with XRCC1 for Lig3␣ stability (Fig.…”

“…One possibility is that Lig1 can fulfill this role, because this DNA ligase is associated with the DNA replication machinery and can conduct BER in vitro (29,35,40). It will be of interest to examine whether the requirement for XRCC1-Lig3␣ interaction during SSBR following oxidative stress exhibits cell cycle specificity, as has been reported for this interaction following ethyl methanesulfonate-induced DNA alkylation (36,48).…”

“…Once again, it is unlikely that this reflects residual interaction between XRCC1 and Lig3␣. This is because interaction with XRCC1 is required to stabilize the DNA ligase in vivo, yet the cellular level of Lig3␣ is no higher in EM9-XH ␦529-633 cells expressing XRCC1 that lack the BRCT II domain than in EM9 cells that lack XRCC1 altogether (36). Consequently, either Lig3␣ can function independently of XRCC1 following oxidative stress or, more likely, another DNA ligase can substitute for Lig3␣ in proliferating CHO cells.…”

Central Role for the XRCC1 BRCT I Domain in Mammalian DNA Single-Strand Break Repair

“…To examine the biological importance of the BRCT I domain, mutations were created that were analogous to those previously shown to disrupt the folding (57) and activity (36,46,47) of the BRCT II domain (Fig. 1A).…”

“…A striking feature of XRCC1 is the presence of two BRCA1 carboxyl-terminal (BRCT) domains, denoted BRCT I and BRCT II, that are located centrally and at the C terminus of this polypeptide, respectively (5,12). The C-terminal domain is responsible for binding and stabilizing Lig3␣ (31,37,47) and is required for SSBR specifically during the G 0 /G 1 phase of the cell cycle (36,46). In contrast, little is known about the role of the central BRCT I domain.…”