DNA sequence context greatly affects the accuracy of bypass across an ultraviolet light 6-4 photoproduct in mammalian cells

Abstract

Translesion DNA synthesis (TLS) is a DNA damage tolerance mechanism carried out by low-fidelity DNA polymerases that bypass DNA lesions, which overcomes replication stalling. Despite the miscoding nature of most common DNA lesions, several of them are bypassed in mammalian cells in a relatively accurate manner, which plays a key role maintaining a low mutation load. Whereas it is generally agreed that TLS across the major UV and sunlight induced DNA lesion, the cyclobutane pyrimidine dimer (CPD), is accurate, there were conflicting reports on whether the same is true for the thymine–thymine pyrimidine–pyrimidone(6-4) ultraviolet light photoproduct (TT6-4PP), which represents the second most common class of UV lesions. Using a TLS assay system based on gapped plasmids carrying site-specific TT6-4PP lesions in defined sequence contexts we show that the DNA sequence context markedly affected both the extent and accuracy of TLS. The sequence exhibiting higher TLS exhibited also higher error-frequency, caused primarily by semi-targeted mutations, at the nearest nucleotides flanking the lesion. Our results resolve the discrepancy reported on TLS across TT6-4PP, and suggest that TLS is more accurate in human cells than in mouse cells.

Eukaryotic Translesion DNA Synthesis on the Leading and Lagging Strands: Unique Detours around the Same Obstacle

“…Furthermore, UV irradiation elicits monoubiquitination of PCNA by Rad6/Rad18 and phosphorylation of pol η, both of which are imperative for pol η-mediated TLS in vivo 34, 198 . However, Polη has an eightfold preference for mis-insertion at (6–4)PPs and pols ζ, ι, and κ all perform mutagenic TLS across CPDs in human cells lacking functional pol η 58, 65, 67, 68 . Thus, DNA-damaging agents and the cellular response they trigger may generically target TLS pols to damaged DNA but selection of the appropriate TLS pol(s) and, hence, the fidelity of DNA damage bypass is ultimately governed by the DNA lesion itself and perhaps the surrounding sequence.…”

“…Thus, the vast majority of (6–4) PPs are repaired prior to an encounter with a moving replisome while most CPD lesions persist into and throughout S-phase. However, both lesions can be accommodated, i.e., “tolerated,” during S-phase by TLS and this process absolutely requires the interaction of TLS pols with PCNA encircling the damaged DNA 58–64 .…”

“…Pols η and ι function in an error-prone manner while pol ζ promotes high fidelity TLS 58, 65 . It should be noted that (6–4) PPs may also be accommodated in lower eukaryotes by an alternative, error-free DDT pathway that is independent of TLS pols.…”

“…UV-induced lesions that persist into S-phase can be accommodated by TLS and this DDT pathway absolutely requires the interaction of TLS pols with PCNA encircling the damaged DNA 58–64 . As discussed above, extensive stretches of ssDNA are generated when UV-induced lesions are encountered by the replisome, especially within the leading strand template, and the aforementioned models for TLS provide options for when these abnormalities are resolved.…”

Y-family DNA polymerase-independent gap-filling translesion synthesis across aristolochic acid-derived adenine adducts in mouse cells

“…This result suggests that the fidelity of TLS can also contribute to the sequence preference for mutation induction as well as the ease of adduct formation and the resistance to nucleotide excision repair. Another study has also reported that sequence contexts contribute to the efficiency and coding property of TLS across a T-T (6–4) photoproduct (60). The mechanism by which neighboring bases influence the property of TLS remains to be determined…”