Highly error-free role of DNA polymerase η in the replicative bypass of UV-induced pyrimidine dimers in mouse and human cells

Abstract

Cyclobutane pyrimidine dimers (CPDs) constitute the most frequent UV-induced DNA photoproduct. However, it has remained unclear how human and other mammalian cells mitigate the mutagenic and carcinogenic potential of CPDs emanating from their replicative bypass. Here, we examine in human cells the roles of translesion synthesis (TLS) DNA polymerases (Pols) in the replicative bypass of a cis-syn TT dimer carried on the leading or the lagging strand DNA template in a plasmid system we have designed, and we determine in mouse cells the frequencies and mutational spectra generated from TLS occurring specifically opposite CPDs formed at TT, TC, and CC dipyrimidine sites. From these studies we draw the following conclusions: (i) TLS makes a very prominent contribution to CPD bypass on both the DNA strands during replication; (ii) Pols , , and provide alternate pathways for TLS opposite CPDs wherein Pols and promote mutagenic TLS opposite CPDs; and (iii) the absence of mutagenic TLS events opposite a cis-syn TT dimer in human cells and opposite CPDs formed at TT, TC, and CC sites in mouse cells that we observe upon the simultaneous knockdown of Pols and implicates a highly error-free role of Pol in TLS opposite CPDs in mammalian cells. Such a remarkably high in vivo fidelity of Pol could not have been anticipated in view of its low intrinsic fidelity. These observations have important bearing on how mammalian cells have adapted to avoid the mutagenic and carcinogenic consequences of exposure to sunlight.DNA damage and repair ͉ translesion synthesis ͉ UV damage U V light induces the formation of two major photoproducts, cis-syn cyclobutane pyrimidine dimers (CPDs) and (6-4) pyrimidine-pyrimidone photoproducts [(6-4) PPs]. Of the two lesions, (6-4) PPs are removed rapidly and efficiently by nucleotide excision repair (NER), whereas the removal of CPDs by NER occurs at a much slower rate (1); hence, CPDs constitute a much more important premutagenic lesion than (6-4) PPs, and it has been estimated that CPDs account for Ϸ80% of UV-induced mutations in mammalian cells (2). Because of their relatively high abundance, slow repair, and the fact that CPDs account for a large majority of UV-induced mutations (3, 4), in this study we determine the roles that human and mouse translesion synthesis (TLS) Pols , , , and play in TLS opposite CPDs and the relative contributions they make to their error-free vs. mutagenic bypass.For TLS studies in human cells, we have designed a duplex plasmid system in which bidirectional replication ensues from a replication origin and proceeds through a site-specific cis-syn TT dimer carried on the leading or the lagging DNA strand template. Studies with this plasmid system in human cells have allowed us to make several unanticipated observations, some of which we note here: (i) opposite a cis-syn TT dimer, TLS makes an important contribution to lesion bypass and is predominantly error-free; (ii) different TLS Pols contribute similarly to lesion bypass on the leading and lagging DNA strands; (iii) Po...

Stability of the human polymerase δ holoenzyme and its implications in lagging strand DNA synthesis

“…Human pol η accurately replicates across thymine-thymine CPDs (TT-CPDs) in vitro and is responsible for the highly errorfree (>90%) bypass of UV-induced CPDs in vivo (53,54). Interestingly, the DNA binding affinity of human pol η alone is not enhanced by the presence of a TT-CPD, and it progressively weakens on extending a P/T junction such that the pol η•DNA complex is destabilized after incorporating only two dNTPs beyond the lesion (55).…”

DNA polymerase η lacking the ubiquitin-binding domain promotes replicative lesion bypass in humans cells

“…The duplex plasmid that carries a cis-syn TT dimer on the leading-strand or the lagging-strand template has been described previously (4). Stably transfected XPV cells were plated in a six-well plate with 80% confluence.…”

“…We have shown previously that Pols η, κ, and ζ provide three alternative pathways for promoting replication through CPDs in human as well as mouse cells, wherein Polη functions in a highly error-free manner and Pols κ and ζ function in a more mutagenic manner (4). Hence the frequency of mutagenic TLS rises in the absence of Polη.…”

“…Biochemical and structural studies have indicated that they function in TLS in a highly specialized manner, providing an efficient and relatively accurate way to synthesize DNA opposite a diverse array of DNA lesions (1). Among TLS Pols, Polη is highly efficient at replicating through UV-induced cyclobutane pyrimidine dimers (CPDs); and surprisingly, in spite of its rather low intrinsic fidelity of 1 × 10 −2 to 3 × 10 −2 (2, 3), Polη promotes highly errorfree replication through CPDs in human cells (4). Inactivation of Polη in humans causes the cancer-prone syndrome, the variant form of xeroderma pigmentosum (XPV) (5,6).…”

Rapid Deamination of Cyclobutane Pyrimidine Dimer Photoproducts at TCG Sites in a Translationally and Rotationally Positioned Nucleosome in Vivo

“…1A) (1)(2)(3)(4)(5). Because C-containing CPDs must deaminate to become mutagenic by the polymerase pathway, factors that accelerate deamination are also expected to enhance C to T mutation frequency.…”