The Polymerase η Translesion Synthesis DNA Polymerase Acts Independently of the Mismatch Repair System To Limit Mutagenesis Caused by 7,8-Dihydro-8-Oxoguanine in Yeast

Abstract

Reactive oxygen species are ubiquitous mutagens that have been linked to both disease and aging. The most studied oxidative lesion is 7,8-dihydro-8-oxoguanine (GO), which is often miscoded during DNA replication, resulting specifically in GC 3 TA transversions. In yeast, the mismatch repair (MMR) system repairs GO ⅐ A mismatches generated during DNA replication, and the polymerase (Pol) translesion synthesis DNA polymerase additionally promotes error-free bypass of GO lesions. It has been suggested that Pol limits GO-associated mutagenesis exclusively through its participation in the filling of MMR-generated gaps that contain GO lesions. In the experiments reported here, the SUP4-o forward-mutation assay was used to monitor GC 3 TA mutation rates in strains defective in MMR (Msh2 or Msh6) and/or in Pol activity. The results clearly demonstrate that Pol can function independently of the MMR system to prevent GO-associated mutations, presumably through preferential insertion of cytosine opposite replication-blocking GO lesions. Furthermore, the Pol-dependent bypass of GO lesions is more efficient on the lagging strand of replication and requires an interaction with proliferating cell nuclear antigen. These studies establish a new paradigm for the prevention of GO-associated mutagenesis in eukaryotes.Eukaryotic genome stability can be compromised by changes at the nucleotide level, alterations in chromosome structure, or changes in chromosome number. Although such changes are responsible for many human diseases, including cancer, a low level of instability is necessary to provide the raw material for evolutionary processes. Changes at the nucleotide level generally occur during replication, either as errors made when copying an undamaged DNA template or during the bypass of DNA lesions. Many types of DNA lesions are due to reactive oxygen species (ROS), which are generated by exposure to physical and chemical mutagens, as well as by normal metabolic processes, such as aerobic respiration (12, 32). Although cells contain multiple antioxidants and other proteins that protect the genome from oxidative damage, ROS have been implicated as causal agents of many diseases and of aging (11,50).The most common oxidized DNA lesion is 7,8-dihydro-8-oxoguanine, which is referred to here as a GO lesion. The mutagenic potential of this lesion is due to miscoding during DNA synthesis, with replicative DNA polymerases usually misinserting adenine across from the lesion to generate GO ⅐ A mispairs and ultimately GC 3 TA transversions (49). Studies examining the crystal structure of T7 DNA polymerase complexed with a GO ⅐ C base pair or a GO ⅐ A mispair indicate the basis of this mutagenic specificity. Whereas the GO ⅐ C structure physically resembles that of a mismatch, the GO ⅐ A mispair structurally resembles a normal Watson-Crick base pair and therefore is likely to escape polymerase-associated proofreading activity (6). A GO-containing nucleotide triphosphate (8-oxo-dGTP) can also be used by DNA polymerases during DNA synthesis,...

DNA Repair Mechanisms and the Bypass of DNA Damage in Saccharomyces cerevisiae

“…How the initiating nicks are targeted specifically to the nascent strand and precisely how the nicked strand is removed remain to be fully elucidated, and differences may exist depending on whether an error arises during leading-vs. lagging-strand synthesis. Indeed, the MMR system more efficiently removes lagging-than leading-strand errors Kow et al 2007;Mudrak et al 2009;Lujan et al 2012). Most genetic and structure-function studies of eukaryotic MMR have been done in yeast, whereas most attempts to biochemically reconstitute the system have utilized purified human proteins.…”

“…No significant involvement of Pol h has been observed in a gap-filling assay (Gibbs et al 2005) or during bypass of genomic AP sites (Otsuka et al 2002b;Auerbach and Demple 2010;Kim et al 2011b). With respect to 8-oxoG, Pol h has a strong preference for inserting C rather than A in vitro McCulloch et al 2009), and there is a strong increase in 8-oxoG-associated mutagenesis upon deletion of the RAD30 gene De Padula et al 2004;Mudrak et al 2009;Van Der Kemp et al 2009). As noted previously, Pol h is part of the yeast GO network (Figure 2).…”

“…The PIP domain is essential for survival following UV irradiation (Haracska et al 2001a) and for errorfree 8-oxoG bypass (Mudrak et al 2009). Pol h additionally has a Ub-binding (UBZ) domain that further facilitates interaction with mono-Ub PCNA and is required for in vivo function (De Padula et al 2004;Parker et al 2007;Pabla et al 2008;Van Der Kemp et al 2009).…”

In Vivo Bypass of 8-oxodG

“…The interplay of TLS and MMR is not completely clear. It was proposed that MMR was responsible for recruiting Pol η for bypass [18] but a detailed study of 8-oxoG bypass and repair concluded that Pol η acted independently of MMR [19]. It appears that monoubiquitination of PCNA is necessary for most TLS and in yeast this step is carried out by the Rad6-Rad18 heterodimer [20], [21].…”

RAD18 gene product of yeast Saccharomyces cerevisiae controls mutagenesis induced by hydrogen peroxide

“…The second barrier to mutagenesis is a correction system for mis pared bases (mismatch repair), which excises adenine from the 8 oxoG:A mispair arising as a result of repli cation of damaged matrix [7]. The third barrier is the Роlη DNA polymerase, which in the process of filling the gaps formed by excising adenine opposite 8 oxoG during mismatch repair or in blocking the replicative complex at the damaged guanine base, inserts cytosine opposite 8 oxoG [17,18]. It is known that Роlη is attracted to the site of matrix lesion through the Rad6-Rad18 dependent PCNA monoubiquitylation [19][20][21].…”