Ribonucleotide incorporation by human DNA polymerase η impacts translesion synthesis and RNase H2 activity

Abstract

Ribonucleotides (rNs) incorporated in the genome by DNA polymerases (Pols) are removed by RNase H2. Cytidine and guanosine preferentially accumulate over the other rNs. Here we show that human Pol η can incorporate cytidine monophosphate (rCMP) opposite guanine, 8-oxo-7,8-dihydroguanine, 8-methyl-2΄-deoxyguanosine and a cisplatin intrastrand guanine crosslink (cis-PtGG), while it cannot bypass a 3-methylcytidine or an abasic site with rNs as substrates. Pol η is also capable of synthesizing polyribonucleotide chains, and its activity is enhanced by its auxiliary factor DNA Pol δ interacting protein 2 (PolDIP2). Human RNase H2 removes cytidine and guanosine less efficiently than the other rNs and incorporation of rCMP opposite DNA lesions further reduces the efficiency of RNase H2. Experiments with XP-V cell extracts indicate Pol η as the major basis of rCMP incorporation opposite cis-PtGG. These results suggest that translesion synthesis by Pol η can contribute to the accumulation of rCMP in the genome, particularly opposite modified guanines.

Ribonucleotide discrimination by translesion synthesis DNA polymerases

“…Eukaryotic cells have two RAD30 paralogs, pol η and pol ι. Interestingly, in the presence of rNTPs, the behavior of human pol η is closer to its human relative, pol ι, rather than to its S. cerevisiae pol η counterpart (Donigan et al , 2014; Donigan et al , 2015; Mentegari et al , 2017; Su et al , 2016). …”

“…On the contrary, the human homolog is much less prone to distinguish the identity of the sugar moiety [Table 1. (Mentegari et al , 2017; Su et al , 2016)]. Human pol η (hpol η) not only efficiently synthesizes DNA/RNA and RNA fragments on undamaged DNA templates, but also does it much more accurately, compared to synthesis in the presence of dNTPs alone.…”

“…Human pol η (hpol η) not only efficiently synthesizes DNA/RNA and RNA fragments on undamaged DNA templates, but also does it much more accurately, compared to synthesis in the presence of dNTPs alone. Furthermore, hpol η displays a similar efficiency and accuracy of rNTP incorporation when replicating various damaged DNAs, such as 8-oxo-G, T-T cyclobutane pyrimidine dimers, 8-methyl-2’-deoxyguanosine, and cisplatin-GG intrastrand crosslink (Mentegari et al , 2017; Su et al , 2016). Based on these findings, it has been suggested that TLS by hpol η can contribute to the accumulation of rNMPs into genomic DNA, and particularly rCMPs paired with modified guanines.…”

“…Based on these findings, it has been suggested that TLS by hpol η can contribute to the accumulation of rNMPs into genomic DNA, and particularly rCMPs paired with modified guanines. The impact of such specificity is especially significant, since human RNase H2 which is responsible for the initiation of ribonucleotide excision repair is least efficient while removing rCMPs and rGMPs and is often inhibited when the base paired with the rNMP is damaged (Mentegari et al , 2017). …”

“…An important contribution into understanding the mechanism regulating the ability of hpol η to incorporate ribonucleotides has been made using the crystal structure of the human DNA polymerase with an incoming ribonucleotide (rC) opposite both an undamaged DNA base (dG) (Figure 3) and an 8-oxodG lesion (Mentegari et al , 2017; Su et al , 2016). It has been shown that pol η scaffolds the incoming rNTP to pair with the complementary template with a significant propeller twist.…”