Although results from yeast suggest that pol η does not confer resistance to ICL by cisplatin [5
], XP-V cells have been reported to be hypersensitive to and impaired in the repair of a number of DNA damaging agents, including ICL-forming agents, suggesting that TLS by pol η is important in lesion tolerance and survival following ICL in humans [17
]. However, most prior studies of pol η and ICLs have been performed utilizing plasmid substrates, rather than analyzing genomic DNA [18
]. We confirmed that loss of pol η renders cells moderately hypersensitive to genomic photoadducts of the psoralen derivative, HMT, under split-dose UVA conditions designed to generate significant quantities of ICL. Further, unlike single dose irradiation of HMT or of angelicin, which generate mostly or exclusively monoadducts, split-dose UVA induces elevated γ-H2AX levels in XP-V cells relative to wild-type GM637 cells, and this induction returns to normal levels when pol η is ectopically expressed in XP-V cells, indicating that pol η is indeed involved in the cellular response to psoralen ICL in genomic DNA.
The observed reduction of γ-H2AX throughout the cell cycle in the presence of pol η in response to ICL also suggests that pol η can mediate a response to ICL or its repair intermediates that are not necessarily at blocked replication forks. Because there is considerable evidence that γ-H2AX is a marker of DSB [21
], one interpretation of our results is that pol η may contribute to suppression of DSB in response to ICL throughout the cell cycle. Although there is evidence from mammalian cell extracts in which replication is likely not occurring that DSB may still be generated at psoralen ICL [8
], the preponderance of reports have shown that ICL induce DSB during replication [12
]. Thus, an alternative interpretation of the data is that the observed γ-H2AX induction, particularly outside of S-phase, is not entirely in response to DSB formation. Recently, it has been reported that following ultraviolet radiation γ-H2AX does not necessarily reflect DSB during the G1 phase of the cell cycle [42
], as well as in growth-arrested cells in G0 [24
]. Interestingly, ICL-forming agents have been reported to generate ssDNA foci that depend on NER, and ssDNA resulting from impaired gap-filling during nucleotide excision repair has recently been reported to be associated with γ-H2AX induction [24
Our results suggest that pol η participates in a psoralen ICL repair pathway that avoids accumulation of DSB or ssDNA intermediates that serve to trigger induction of γ-H2AX (). While both DSB and ssDNA have been identified as potential intermediate products of replication arrested at a DNA lesion and likely account for a γ-H2AX signal during S-phase, ssDNA associated with incomplete NER may account for γ-H2AX generated during other phases of the cell cycle. NER may operate to uncouple one strand of the ICL, followed by pol η-mediated TLS using the remaining intact strand as a template, as has been generally proposed for other polymerases previously [15
]. While a role for gap-filling during NER by pol η at lesions outside of a replication fork has not been specifically described, polymerase κ, another bypass polymerase in the Y-family to which pol η also belongs, has been proposed to function in this role in NER [44
]. Loss of NER may direct more ICL into another pathway, specifically dependent on XPF/ERCC1 but not NER, that processes the lesions to DSB [21
]. Such a model also accounts for the extreme hypersensitivity of cells deficient in XPF or ERCC1 which appear to be involved in both DSB-forming and NER/TLS repair pathways [21
Model for psoralen ICL repair pathways involving polymerase η and their relationship to γ-H2AX.
An alternate but not mutually exclusive possibility consistent with our results is that pol η functions in a separate pathway downstream of ICL-induced γ-H2AX associated with DSB, and that loss of pol η leads to an accumulation of irreparable DSB, leading to enhanced γ-H2AX levels (). Recently, it has been proposed that, following DSB formation by a blocked replication fork at an ICL, TLS occurs as a necessary prelude for homologous recombination [45
], and pol η could be the TLS polymerase involved in this model. In addition, pol η has also been reported to be involved in homologous recombination with a polymerase activity independent of its TLS functions [46
] and could function in recombinational repair of DSB resulting from ICL.
The relative importance of pol η and other TLS polymerases in ICL repair is unclear. Recent studies have suggested that polymerase ζ is the predominant determinant of ICL repair [14
]. However, in yeast, REV3 activity in ICL repair was restricted exclusively to the G1 phase of the cell cycle, leaving open the possibility that other TLS polymerases might fulfill similar roles in other phases of the cell cycle [15
]. Our data suggest that pol η may have a more general role in the response to ICL throughout the cell cycle, and may complement the more restricted activity of polymerase ζ. In addition, pol η and pol ζ may function cooperatively in TLS at an ICL. Pol η is unique in its ability to perform translesion synthesis opposite cyclobutane pyrmidine dimers presumably due to its extremely open catalytic site. However, pol η often has difficulty extending past bulkier or more distorting DNA lesions [48
]. In contrast, pol ζ is extremely poor in replicating opposite a wide variety of DNA lesions, and appears to function best as an extender of 3’ ends opposite a lesion. Thus, both polymerases may be necessary to optimally bypass the unusual structure resulting from an initial ICL uncoupling.