DNA Replication, Repair, and Mutagenesis In Eukaryotic And Prokaryotic Cells

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

Scientists in the Section on DNA Replication, Repair and Mutagenesis (SDRRM) study the mechanisms by which mutations are introduced into DNA. These studies have traditionally spanned the evolutionary spectrum and include studies in bacteria, archaea and eukaryotes and involve collaborations with scientists around the world. Strand specificity of Ribonucleotide Excision Repair in E.coli In Escherichia coli, replication of both strands of genomic DNA is carried out by a single replicase DNA polymerase III holoenzyme (pol III HE). However, in certain genetic backgrounds, the low-fidelity TLS polymerase, DNA polymerase V (pol V) gains access to undamaged genomic DNA where it promotes elevated levels of spontaneous mutagenesis preferentially on the lagging strand. As part of a collaboration with scientists at the Polish Academy of Sciences in Warsaw, Poland, we employed active site mutants of pol III (pol III alpha_S759N) and pol V (pol V_Y11A) to analyze ribonucleotide incorporation and removal from the E. coli chromosome on a genome-wide scale under conditions of normal replication, as well as SOS induction. Using a variety of methods tuned to the specific properties of these polymerases (e.g., analysis of lacI mutational spectra, lacZ reversion assay, HydEn-seq, and alkaline gel electrophoresis), we presented evidence that repair of ribonucleotides from both DNA strands in E. coli is unequal. While RNase HII plays a primary role in leading-strand Ribonucleotide Excision Repair (RER), the lagging strand is subject to other repair systems (RNase HI and under conditions of SOS activation also Nucleotide Excision Repair). Importantly, we suggested that RNase HI activity can also influence the repair of single ribonucleotides incorporated by the replicase pol III HE into the lagging strand. Identification of an inhibitor of LexA cleavage As antibiotic resistance has become more prevalent, the social and economic impacts are increasingly pressing. Indeed, bacteria have developed the SOS response which facilitates the evolution of resistance under genotoxic stress. The transcriptional repressor, LexA, plays a key role in this response. Mutation of LexA to a non-cleavable form that prevents the induction of the SOS response sensitizes bacteria to antibiotics. Achieving the same inhibition of proteolysis with small molecules also increases antibiotic susceptibility and reduces drug resistance acquisition. Previous attempts at developing inhibitors have investigated 1,2,3-triazole molecules binding to the hydrophobic cleft, and boronic acids that covalently bound to Ser-119. Neither of these resulted in any molecules going to preclinical trials. In collaboration with scientists at the Queensland Institute of Technology in Brisbane, Australia, we found that the cleavage site region (CSR) of the LexA protein is a classical Type II beta-turn, and that published 1,2,3-triazole compounds mimic the beta-turn. Based upon this, we took a dual approach to the identification of a novel proteolytic inhibitor. Generic covalent molecule libraries and a -turn mimetic library were docked to the LexA C-terminal domain using molecular modelling methods in FlexX and CovDock. The 133 highest scoring molecules were screened for their ability to inhibit LexA cleavage under alkaline conditions and the top molecules were then tested using a RecA-mediated counter assay. This research led to the discovery of an electrophilic serine warhead that can inhibit LexA proteolysis, reacting with Ser-119 via a nitrile moiety. Our studies therefore present a starting point for hit-to-lead optimization, which could lead to inhibition of the SOS response and prevent the acquisition of antibiotic resistance. Characterization of the mycobacterial mutasome A DNA damage-inducible mutagenic gene cassette has been implicated in the emergence of drug resistance in Mycobacterium tuberculosis during anti-tuberculosis (TB) chemotherapy. However, the molecular composition and operation of the encoded mycobacterial mutasome minimally comprising DnaE2 polymerase and ImuA and ImuB accessory proteins remain elusive. As part of a large international collaboration led by Digby Warner at the University of Cape Town, South Africa, we exposure mycobacteria to DNA damaging agents and observed that DnaE2 and ImuB co-localize with the DNA polymerase III beta subunit (beta clamp) in distinct intracellular foci. Notably, genetic inactivation of the mutasome in an imuB mutant containing a disrupted beta clamp-binding motif abolishes ImuB-beta clamp focus formation, a phenotype recapitulated pharmacologically by treating bacilli with griselimycin and in biochemical assays in which this beta clamp-binding antibiotic collapses pre-formed ImuB-beta clamp complexes. These observations established the essentiality of the ImuB-beta clamp interaction for mutagenic DNA repair in mycobacteria and identifies the mutasome as a target for adjunctive therapeutics designed to protect anti-TB drugs against emerging resistance.