Ribonucleotide discrimination by translesion synthesis DNA polymerases

Abstract

The well-being of all living organisms relies on the accurate duplication of their genomes. This is usually achieved by highly elaborate replicase complexes which ensure that this task is accomplished timely and efficiently. However, cells often must resort to the help of various additional "specialized" DNA polymerases that gain access to genomic DNA when replication fork progression is hindered. One such specialized polymerase family consists of the so-called "translesion synthesis" (TLS) polymerases; enzymes that have evolved to replicate damaged DNA. To fulfill their main cellular mission, TLS polymerases often must sacrifice precision when selecting nucleotide substrates. Low base-substitution fidelity is a well-documented inherent property of these enzymes. However, incorrect nucleotide substrates are not only those which do not comply with Watson-Crick base complementarity, but also those whose sugar moiety is incorrect. Does relaxed base-selectivity automatically mean that the TLS polymerases are unable to efficiently discriminate between ribonucleoside triphosphates and deoxyribonucleoside triphosphates that differ by only a single atom? Which strategies do TLS polymerases employ to select suitable nucleotide substrates? In this review, we will collate and summarize data accumulated over the past decade from biochemical and structural studies, which aim to answer these questions.

Keywords: DNA polymerase; mutant polymerases; replicative bypass; ribonucleotide incorporation; steric gate; translesion DNA synthesis.

Figure 1.. Chemical structure of double-stranded DNA formed by the covalently linked sugar rings (shown in blue), phosphate groups (yellow) and nitrogenous bases.

The segment shown on the diagram consists of three cytosine (green) / guanine (pink) Watson-Crick base pairs where two deoxycytidines (dC) are replaced with either cytidine (rC) or 1-β-D-arabinofuranosylcytosine (araC). The 2′-OH groups (highlighted in red) of the ribonucleotide and arabinofuranoside are on the opposite sides of the plane of the sugar. A color version of the figure is available online.

Figure 2.. Multiple sequence alignments of the amino acid region involved in sugar discrimination in TLS DNA polymerases.

(A) Sequence alignments for TLS polymerases from A-, B, X, and Y families. The highly conserved single amino acid residue within each sequence responsible for the steric exclusion of ribonucleotides is shown in red letters over a yellow background and is indicated by the red star below the alignment. The unusual steric gate Gly in pol μ is indicated in green over a red background. The unconventional steric gate residues [I, L, Q and V] found in some DinB polymerases from Actinobacteria are shown in yellow letters over a red background. Almost invariant amino acid [F] juxtaposed to the steric gate residue of Y-family polymerases is shown in blue letters over a green background and is indicated by the blue star below the alignment. The numbers indicate the amino acid position of the first residue shown and are relative to the N-terminus for each polymerase. The species abbreviations are as follows: Ec, Escherichia coli; Ms, Mycobacterium smegmatis; Mm, Microbacterium mangrovi; GA, Gordonia araii; Cf, Cellulomonas fimi; Sa, Sulfolobus acidocaldarius; Ss, Sulfolobus solfataricus; Sc, Saccharomyces cerevisiae; Hs, Homo sapiens. (B) Steric gate polymorphism among actinobacterial DinB homologs. A sequence logo for the fragment of DNA polymerase IV from Actinobacteria consisting of steric gate residue and three flanking residues on each side was created using Weblogo program (Crooks et al., 2004; Schneider and Stephens, 1990). The original sequence set consisted of 3759 actinobacteria sequences from Uniprot depository (including computationally translated sequences from TrEMBL database), predicted by InterPro signature IPR022880 as belonging to DNA polymerase IV family (retrieved on September 3, 2017, http://www.ebi.ac.uk/interpro/entry/IPR022880) (Finn et al., 2017). Sequences exceeding the threshold of 90% identity were removed, resulting in the smaller set of 1550 proteins. Multiple sequence alignment was performed using Kalign program (Lassmann et al., 2009). Sequences poorly aligned or with the gaps in the steric gate region were removed, which left 1347 proteins that were used for the sequence logo construction. Amino acids are color-coded according to their molecular weight (MW). Steric gate position in the wild type DNA polymerases are most often occupied by bulky residues (shown in red). Amino acids with lower MW identified in DinB homologs from Actinobacteria are shown in yellow. Amino acids containing the smallest side chains (shown in green) are often used to replace the steric gate residues in recombinant mutant polymerases. We thank William Taft and Iosif Vaisman (George Mason University) for creating the Dpo4 alignments and logo. A color version of the figure is available online.

Figure 3.. Insertion of an incoming rCTP (shown in yellow) opposite template dG (shown in green) by human pol η (light blue).

The F18 steric gate residue of hpol η, the 2’ OH group of the sugar ring in rCTP, and Ca²⁺ ions (blue) are indicated. (PDB ID 5EWE) (Su et al., 2016). For details see section 3.2. A color version of the figure is available online.

Figure 4.. Structural models of ribonucleotide exclusion by wild type and steric gate variants of E. coli DNA pol V.

Models of wild type and mutant UmuC inserting dATP (shown in yellow) or rATP (blue) opposite a template T (orange) are based on the structure of human DNA pol η (PDB 3MR3). Comparison of the structures of wild type UmuC incorporating dATP (A) and rATP (B) provides an explanation of the unusually low sugar selectivity of pol V. The steric gate residue, Y11, of the wild type enzyme is characterized by increased flexibility. Rotation of the Y11A benzene ring (alternate positions shown in pink and grey) allows for the accommodation of the hydroxyl group at the 2’ position of the nucleoside (indicated in red). (C) Substitution of the highly conserved F10 residue (shown in grey) with leucine (blue) results in dramatic increase in sugar selectivity because the side chain of leucine presses on the benzene ring of Y11 positioning it closer to the C2 position of the sugar moiety of the incoming nucleotide thereby favoring deoxyribonucleotide (dATP) selection and preventing ribonucleotide incorporation. (D) Substitution of the steric gate Y11 with alanine (shown in green) containing smaller side chain creates a void in the active site of UmuC thus relaxing the minor groove alignment for correct Watson-Crick base pair and facilitating accommodation of the 2’-OH group of the incoming nucleotide (shown for ATP). Adapted from (Vaisman et al., 2012a), with permission. A color version of the figure is available online.