The eChIP approach allows DNA damage processing/signaling factors to be directly linked to a defined lesion and detected with significantly improved sensitivity and specificity. Unlike many other viral replication origins, EBV ori possesses a defined replication initiation site with a virtual unidirectionality (Gahn and Schildkraut, 1989
) that allows the generation of a crosslink-blocked replication fork at a precise location with enhanced synchrony. This is in contrast to mammalian genomes which lack defined replication origins. The replication elongation complex on the eChIP substrate consists of exclusively endogenous replication factors, enabling the eChIP system to faithfully reflect mammalian DNA replication.
The eChIP system allowed us to uncover distinct recruitment patterns among canonical FA and breast cancer-related FA proteins at the site of a defined crosslink. Our results provide the direct molecular evidence that the FA proteins, including the BRCA-related proteins, are recruited to the sites of DNA interstrand crosslinks. The presence of the FA core complex at a crosslinked site and its indispensable role in the recruitment of FANCD2 suggests that the FA core complex recognize crosslinked DNA or its processed intermediates. Given that the enrichment of both the core and the I/D2 complex occurs in nonreplicative crosslinked substrate, it is likely that FANCD2 ubiquitination/activation can be achieved in the absence of blocked replication. Indeed, we found that the introduction of crosslinked substrate, but not the unmodified control plasmid, resulted in FANCD2 ubiquitination in 293-EBNA as well as in 293 cells.
In G1/G0 cells where DNA replication is dormant, interstrand crosslinks are removed by a recombination-independent mutagenic pathway utilizing the combined actions of nucleotide excision repair and lesion bypass DNA synthesis (Sarkar et al., 2006
; Wang et al., 2001
; Zheng et al., 2003
). Interestingly, recruitment of FANCA exhibited a strong dependence on XPC and XPA (), both critical for the recognition of DNA crosslinks. Thus, prior recognition of the crosslinks by NER appears to precede the function of the FA core complex during recombination-independent ICL repair. We also observed visible losses of FANCA enrichment in EBV-immortalized XPA and XPC mutants (data not shown), most likely from the nonreplicating substrates subjected to recombination-independent repair. This results further supports that the FA core and I/D2 complexes may play a role in recognizing processed intermediate of recombination-independent mechanism. It is possible that a ubiquitinated FANC I/D2 complex may be required for the loading of a lesion bypass polymerase for gap synthesis. Consistent with this, FANCD2 mutant cells were found in our study to be partially defective in recombination-independent crosslink repair, which can be caused by a defect in the postulated FANC core/ID function of loading lesion bypass polymerase(s). In contrast, we found that loss of FANCJ led to enhanced recombination-independent crosslink repair. This result may reflect, albeit indirectly, a passive competition between the NER/lesion bypass-based homology-independent crosslink repair and the homology-dependent crosslink repair pathways, which presumably is defective in BRCA-related FA mutants (Litman et al., 2005
). It is also conceivable that the Brca-related FA proteins are involved in re-establishing the collapsed replication forks after crosslink removal (), which is likely reflected by the prolonged G2/M arrest in FANCJ mutant cells treated with mitomycin C (Fig. S3
Collectively, it appears that FA proteins are involved in two distinct or branched processes dealing with crosslinks. Cell biology studies suggested that crosslink-induced BRCA2 and FANCD2 focus formation were independent of each other (Ohashi et al., 2005
), although the exact role of replication in this process was not examined. The fact that the recruitment of FANCJ and FANCN does not require the FANC core complex further indicates at the molecular level that a stalled replication fork per se
might be sufficient for the recruitment of recombination-related FANC proteins. Despite the similarities between the phenotypes of patients bearing the FA alleles of BACH1, PALB2, and BRCA2 and patients with mutations in the canonical FA genes, there is a shortage of evidence that these two branches directly work together. Our data indicate that they possess independent functions, at least with respect to their abilities to be recruited to sites of damage. This functional deviation between the canonical FA proteins and the BRCA- and homologous recombination-related FA proteins is perhaps reflected also by their distinct tumor spectrum with the latter exhibiting higher prevalence of breast cancer(Wagner et al., 2004
). Thus, it appears that the complexities of the FA pathway may resemble those of the xeroderma pigmentosum (XP) proteins whose function encompass three distinct aspects of UV damage response that include lesion bypass synthesis, nucleotide excision repair, and damage signaling via ubiquitination.