Involvement of Nucleotide Excision Repair in a Recombination-Independent and Error-Prone

Pathway of DNA Interstrand Cross-Link Repair

Alkylating agents were among the first compounds found to be efficacious in cancer therapy
and remain important components of many modern chemotherapeutic regimens (38). Many
members of this class of drugs have bifunctional groups that can react with both strands of
the DNA helix and thus form interstrand and intrastrand cross-links. As profound blocks for
both transcription and DNA replication, interstrand cross-links (ICLs) appear to represent the
primary cytotoxic lesion induced by most bifunctional alkylating agents.

Repair of DNA ICLs has been studied extensively in Escherichia coli (11, 12). Both genetic
and biochemical evidence has established a combined nucleotide excision repair (NER)-
recombination mechanism for the error-free repair of ICLs, in which the gap, created by the
Uvr(A)BC excinuclease, is repaired through recA-mediated recombination with a lesion-free
homologous chromosome as the donor (10, 37, 41). Although the NER-recombination
pathway appears to be the primary mechanism of cross-link repair in E. coli, recent evidence
has suggested a recombination-independent pathway for cross-link repair in which the gap
created by the uvr(A)BC excinuclease is repaired by translesion bypass in order to circumvent
a deficiency in recombination or a lack of homologous donor sequences (4, 5). In the budding
yeast Saccharomyces cerevisiae, members of the RAD52 epistasis group exhibit
hypersensitivity to cross-linking agents and ionizing radiation, indicating that repair of ICLs
requires homologous recombination. Members of the RAD3 epistasis group, which are
deficient in NER, are also highly sensitive to cross-link damage (21, 29, 36). Taken together,
these observations support a combined NER-recombination mechanism for ICL repair in
lower eukaryotes. As is the case in E. coli, recombination-independent mechanisms may also
play a role in cross-link removal in eukaryotes. The yeast pso1 mutant is characterized by
hypersensitivity to psoralen-induced ICLs (19), and genetic analysis has demonstrated
allelism between the pso1-1 mutant and the rev3-1 mutant (9). The REV3 gene encodes the
catalytic subunit of yeast translesion DNA polymerase ζ whose function is required for
induced mutagenesis (30, 33). A more recent study has indicated that rev3p is important for
the processing of ICL repair intermediates in nonreplicating cells, further substantiating a
lesion bypass-based recombination-independent mechanism of ICL repair in yeast (28). A
plausible role for the rev3p-polymerase ζ in cross-link repair is the translesion synthesis past
the lesion upon the uncoupling of a cross-link.

In mammals, mechanisms of ICL repair are largely unknown. The combined NER-
recombination model does not appear to be the major mechanism of repair since the majority
of NER mutants exhibit only mild sensitivity to cross-linking agents, although mutants
defective in either ERCC1 or XPF do exhibit extreme sensitivity (2, 20). However, several
lines of evidence have suggested the involvement of homologous recombination in ICL repair
(39). In vivo, elevated levels of sister chromosome exchange induced by bifunctional
alkylating agents have been well documented and connected to the repair of ICLs (7, 14, 25).
Using a triplex-mediated psoralen cross-link as the model damage, intramolecular
homologous recombination has been reported to occur through both nonconservative single-
strand annealing and conservative reciprocal exchange pathways (15, 16). Recently, more
convincing evidence of recombinational repair of ICLs has emerged through the
characterization of two hamster mutants, irs1 and irs1SF, both of which exhibit extreme
sensitivity to cross-linking agents (17, 24). Both the XRCC2 and the XRCC3 genes, which
complement the repair deficiency of irs1 and irs1SF mutants, respectively, exhibit structural
similarity to the hRad51 recombinase family (27). Studies by Johnson et al. (23) and Pierce et
al. (34) have indicated that both cell lines harbor a defect in homologous recombination
marked by a severe reduction in gene conversion activity. Moreover, RAD54-deficient mouse
embryonic stem cells are also hypersensitive to mitomycin C as an apparent result of reduced
conservative homologous recombination (14). In addition, Li et al. (26) have shown, in vitro,
that the presence of an ICL in a plasmid substrate stimulates repair synthesis in mammalian
cell extracts and that this stimulated synthesis is also observed in an undamaged plasmid
coincubated in the same extract, suggesting that ICLs can induce recombinational repair
synthesis. Taken together, these results provide substantial evidence that recombination
factors participate in ICL repair and are likely to be involved in a major pathway of ICL
removal.

To investigate the mechanisms by which ICLs are repaired in mammalian cells in the absence
of homologous donor sequences, we employed a gene reactivation assay in which a single
defined psoralen ICL was introduced into a reporter plasmid in order to block transcription of
the reporter gene. Consequently, expression of the reporter gene became dependent upon
removal of the ICL. We report here that repair of the ICL present in the plasmid substrate was
observed in wild-type cells and that mutants defective in NER were deficient in reactivation,
while mutants defective in homologous recombination were not. Rescue and sequencing of
repaired plasmids indicated a high rate of mutagenesis at the site of the psoralen cross-link.
These results indicate that a recombination-independent, but error-prone, pathway of ICL
repair exists in mammalian cells.