A single amino acid governs enhanced activity of DinB DNA polymerases on damaged templates

Abstract

Translesion synthesis (TLS) by Y-family DNA polymerases is a chief mechanism of DNA damage tolerance. Such TLS can be accurate or error-prone, as it is for bypass of a cyclobutane pyrimidine dimer by DNA polymerase eta (XP-V or Rad30) or bypass of a (6-4) TT photoproduct by DNA polymerase V (UmuD'2C), respectively. Although DinB is the only Y-family DNA polymerase conserved among all domains of life, the biological rationale for this striking conservation has remained enigmatic. Here we report that the Escherichia coli dinB gene is required for resistance to some DNA-damaging agents that form adducts at the N2-position of deoxyguanosine (dG). We show that DinB (DNA polymerase IV) catalyses accurate TLS over one such N2-dG adduct (N2-furfuryl-dG), and that DinB and its mammalian orthologue, DNA polymerase kappa, insert deoxycytidine (dC) opposite N2-furfuryl-dG with 10-15-fold greater catalytic proficiency than opposite undamaged dG. We also show that mutating a single amino acid, the 'steric gate' residue of DinB (Phe13 --> Val) and that of its archaeal homologue Dbh (Phe12 --> Ala), separates the abilities of these enzymes to perform TLS over N2-dG adducts from their abilities to replicate an undamaged template. We propose that DinB and its orthologues are specialized to catalyse relatively accurate TLS over some N2-dG adducts that are ubiquitous in nature, that lesion bypass occurs more efficiently than synthesis on undamaged DNA, and that this specificity may be achieved at least in part through a lesion-induced conformational change.

Proficient and Accurate Bypass of Persistent DNA Lesions by DinB DNA Polymerases

“…Although deletion of either gene does not alter the frequency of spontaneous mutation, 7,17 it results in an increase in the frequency of mutagenesis induced by certain DNA damaging agents. 11,20 Such behavior is analogously observed with respect to UV-induced mutation in human cells deficient in pol h function, which replicates relatively accurately over thymine-thymine cyclobutane dimers. 30,31 Substrate specificity also appears to be conserved between DinB and pol k. Both enzymes are able to bypass N 2 -B[a]P-adducted template G. 20,32,33 Moreover, they each display a striking 10-15 fold increased activity on a template N 2 -furfuryl-dG relative to undamaged DNA.…”

“…23,54 Our recent discovery of the sensitivity of a DdinB strain to two DNA damaging agents that produce adducts at the N 2 position of dG, nitrofurazone (NFZ) and 4-nitroquinoline 1-oxide (4-NQO), has allowed us to determine whether dinB clearly falls into one of these categories. 11 DinB catalytic function is clearly required for resistance to these agents, but deletion strains show either the same or increased induced mutation frequency relative to the wild-type when treated with either agent. 11 These observations, taken together with the fact that a chromosomal deletion does not alter either spontaneous or induced mutagenesis suggest that dinB is largely anti-mutagenic under many circumstances.…”

“…2), has recently been elucidated. [8][9][10][11][12] Although identified in the first functional screen for transcripts upregulated upon DNA damage in Escherichia coli, 13 an absence of striking and genetically tractable phenotypes, particularly relative to its homologs, resulted in comparatively few insights into DinB function. Here we will summarize the data regarding DinB/pol k dependent mutagenesis and propose a revised view of its function as a specialized DNA polymerase with mutagenic potential that arises along with its unique substrate specificity.…”

“…30,31 Substrate specificity also appears to be conserved between DinB and pol k. Both enzymes are able to bypass N 2 -B[a]P-adducted template G. 20,32,33 Moreover, they each display a striking 10-15 fold increased activity on a template N 2 -furfuryl-dG relative to undamaged DNA. 11 Increased proficiency is also displayed by pol h replicating its cognate substrate T-T cyclobutane pyrimidine dimers. 34 Curiously, both DinB and pol k proficiently extend from a variety of lesions and mismatched primer ends in addition to their insertion specificities.…”

“…34 Curiously, both DinB and pol k proficiently extend from a variety of lesions and mismatched primer ends in addition to their insertion specificities. 8,11,[35][36][37][38][39] It has been suggested, at least for pol k, that this property reflects a separate role in the extension step of TLS. 35 …”