We report here that XRCC1 co-localizes with PCNA replication-specific foci, that these two proteins co-purify from human whole cell extracts and that XRCC1 physically interacts with PCNA in vitro
via residues within 166–310 of XRCC1. Our finding brings the total number of XRCC1-interacting protein partners to at least eight (Fig. A and legend). While XRCC1 lacks a consensus PCNA interaction sequence (i.e. the PIP- or KA-box), other proteins without a consensus motif (e.g. Gadd45 and CAF1) have also been found to associate with PCNA (30
). Moreover, the PCNA-interacting region found here (Fig. A) overlaps the portion of XRCC1 shown to interact with APE1 and OGG1 (8
). Thus, elaborate mechanisms presumably exist to modulate the interactions of these various repair and regulatory proteins.
Figure 5 (A) Schematic of XRCC1 interacting regions and protein partners. Thus far, at least eight proteins (see text for details), including PCNA described in this work, have been found to directly interact with XRCC1. The regions that are responsible for these (more ...)
Both Caldecott and colleagues (15
) and Kubota and Horiuchi (16
) have suggested an S phase-specific role for XRCC1. In fact (as shown here in Fig. ), Taylor et al
), using indirect immunofluorescence, observed S phase-specific XRCC1 foci, which they suggest are connected to RAD51 and DNA recombination. Our results indicate that such XRCC1 foci are associated with replication factories. Since Rad51 nuclear foci are seen exclusively in the S phase of undamaged human cells (38
), the data in total may suggest that replication, repair and recombination are strategically intertwined. We propose that the S phase-specific XRCC1 foci reflect PCNA-directed sequestration of XRCC1 into strand break repair complexes that are associated with translocating DNA replication machines (Fig. B).
While we cannot exclude a role for XRCC1 in replication reinitiation, particularly following DNA damage induction (16
), our ‘replication-coupled repair’ model is consistent with the observation that EM9 cells do not exhibit hypersensitivity to hydroxyurea, an agent used to evaluate the contribution of genetic factors to replication restart (H.-K. Wong and D.M. Wilson III, unpublished observations). In other words, XRCC1 does not appear to maintain a universal role in replication reinitiation. Moreover, since evidence suggests that XRCC1 is not a major participate in long patch, PCNA-dependent BER events (39
), the XRCC1–PCNA foci seen here likely do not represent sites of ‘conventional’ long patch BER. While there is evidence that PCNA co-localizes to sites of atypical SSBs following DNA damage induction (26
), these findings are not directly relevant to our studies, where exogenous DNA-damaging agent exposures were not employed. Nonetheless, they may suggest a cooperative role between XRCC1 and PCNA in certain SSBR processes outside of DNA replication.
A role for XRCC1 in specifically coordinating repair and replication is supported by the facts that XRCC1 mutant cells (i) exhibit an increased doubling time (from 13 to 16 h) (1
) and (ii) display, as a hallmark, markedly elevated SCEs (40
). In particular, spontaneous or EMS-induced SCEs are increased 7- to 12-fold in EM9 cells relative to their wild-type counterparts (1
). While the precise molecular mechanism for SCEs is unclear, this genetic outcome is thought to result from homologous recombination repair of SSBs converted to DSBs upon replication fork collapse (42
). PARP-1, for instance, has been shown to suppress SCEs by promoting efficient repair as opposed to regulating homologous recombination (43
), and this protein, like XRCC1 (data within), has been linked to chromosome replication (44
). On the other hand, POLβ–/–
cells, which are also presumably defective in nick/gap DNA repair, exhibit no increase in spontaneous SCEs and only a mild (2-fold) increase in SCEs upon exposure to the alkylating agent MMS (47
). Thus, the data in total suggest (i) unique functions for XRCC1 and PARP-1 in strand break repair, which we propose to be coordination of repair with replication, and (ii) that polymerases other than POLβ may function in XRCC1-associated SSBR during S phase.
Published data clearly indicate distinct roles for XRCC1 in the G1
and S phases of the cell cycle (15
). As described above, we propose a model (Fig. B) where in S phase XRCC1 specifically facilitates efficient SSBR through its interaction with PCNA (and progressing replication factories), prior to DNA replication arrest, fork collapse and DSB formation. In G1
, XRCC1 likely functions to coordinate direct BER/SSBR events (perhaps represented by the non-S phase foci seen in Fig. B). In particular, following PARP-1 relocalization and recruitment of XRCC1 to the strand break (26
), XRCC1 operates to organize and ‘scaffold’ proteins such as POLβ, PNK and DNA ligase IIIα until repair is complete. Notably, among the small set of BER-related proteins analyzed here, the strongest in vitro
XRCC1 interactor was POLβ (Fig. ). Since we employed only recombinant proteins purified from bacteria, which do not contain eukaryotic-like post-translational modifications, this finding may suggest that XRCC1 is largely unmodified in G1
and, in this state, more proficient at interacting with POLβ, and potentially DNA ligase IIIα, to mediate nick and gap DNA repair. Whether XRCC1 undergoes post-translational modification upon entry into S phase or following exposure to a DNA-damaging agent that alters its ability (or affinity) to specifically interact with proteins in repair, replication and/or recombination (such as PARP-1, PCNA and RAD51) awaits further investigation. Importantly, evidence exists for phosphorylation of the XRCC1 polypeptide (16