Abstract
The spontaneous depurination of genomic DNA occurs frequently and generates apurinic/pyrimidinic (AP) site damage that is mutagenic or lethal to cells. Error-prone DNA polymerases are specifically responsible for the translesion synthesis (TLS) of specific DNA damage, such as AP site damage, generally with relatively low fidelity. The Y-family DNA polymerases are the main error-prone DNA polymerases, and they employ three mechanisms to perform TLS, including template-skipping, dNTP-stabilized misalignment, and misincorporation-misalignment. The bypass mechanism of the dinB homolog (Dbh), an archaeal Y-family DNA polymerase from Sulfolobus acidocaldarius, is unclear and needs to be confirmed. In this study, we show that the Dbh primarily uses template skipping accompanied by dNTP-stabilized misalignment to bypass AP site analogs, and the incorporation of the first nucleotide across the AP site is the most difficult. Furthermore, based on the reported crystal structures, we confirmed that three conserved residues (Y249, R333, and I295) in the little finger (LF) domain and residue K78 in the palm subdomain of the catalytic core domain are very important for TLS. These results deepen our understanding of how archaeal Y-family DNA polymerases deal with intracellular AP site damage and provide a biochemical basis for elucidating the intracellular function of these polymerases.
Highlights
Each organism in the three kingdoms of bacteria, archaea, and eukaryotes possesses more than one DNA polymerase for genome replication and DNA damage repair
Our results showed that a faster and full-length extension occurred for four correctly paired 3 base pairs of A/T or C/G (Figure 1), indicating that the dinB homolog (Dbh) mainly uses template skipping to bypass the spacers for translesion synthesis (TLS)
The skipping of the spacer and primer extension by the Dbh became more difficult (Figure 1B). These results indicated that the Dbh mainly performed TLS using a template skipping mechanism for bypassing spacers and that the correct pairing of the 3 base pairs promoted spacer skipping and the TLS efficiency
Summary
Each organism in the three kingdoms of bacteria, archaea, and eukaryotes possesses more than one DNA polymerase for genome replication and DNA damage repair. TLS DNA polymerases lack a 3 to 5 proofreading activity and synthesize DNA in an error-prone manner. Both Y- and X-family DNA polymerases are error-prone, but only the former participates in the bypass of various damages [7,8]. In addition to Y-family DNA polymerases, the error-prone replicative DNA polymerase DnaE (belonging to the C-family) was found to be able to bypass severely damaged DNA, such as AP sites, and might participate in TLS in vivo [9]. Based on the sequence similarity, Y-family DNA polymerases are divided into six groups: the DinB (pol IV) and UmuDC subfamilies (pol V) of prokaryotes, and the Rad30A (pol η), Rad30B (pol ι), DinB1 (pol κ), and Rev subfamilies of eukaryotes [8,10]
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