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. The Y-family DNA polymerases are responsible for copying damaged DNA during DNA replication in a process called translesion synthesis (TLS). These enzymes are highly specialized in order to accommodate different structural DNA distortions caused by a wide variety of DNA lesions. The Y-family is divided into six phylogenetically distinct subfamilies: two UmuC (pol V) branches; Rad30A/POLH (pol eta); Rad30B/POLI (pol iota); and DinB (pol IV, Dpo4, pol kappa); and Rev1. Across the different domains of life, Y-family polymerase subfamilies are found in various combinations. For example, UmuC orthologs are only detected in Gram-positive and gram-negative bacteria, whereas Rev1 and Rad30A/B orthologs are only detected in eukaryotes. The DinB subfamily is the most evolutionarily conserved, having members scattered throughout all three domains of life from unicellular bacteria to humans. However, differences in the distribution of Y-family DNA pols are present within each kingdom. For example, the eukaryote Saccharomyces cerevisiae (S. cerevisiae) contains neither a pol iota nor a pol kappa gene. Indeed, it was originally assumed that pol iota was expressed only in higher eukaryotes. However, next generation whole genome sequencing has revealed that pol iota orthologs are actually distributed throughout the whole Eukaryota domain. One example is the thermophilic fungus, Thermomyces lanuginosus (T. lanuginosus) which possesses all four eukaryotic Y-family subfamilies much like humans, in contrast to its fungal relatives, S. cerevisiae and Schizosaccharomyces pombe (S. pombe). Is there logic in such seemingly random distribution of pol iota? Using phylogenetic analysis and comparing the biochemical characterization of Y-family pols from different species, we hoped to shed some light on this question. To do so, we described the identification, purification and characterization of thermostable eukaryotic orthologues of pol eta, pol iota, pol kappa, and Rev1 from T. lanuginosus. Biochemical characterization of TLS DNA pols , , , and Rev1 included determination of the enzymes fidelity, processivity, thermostability, metal ion requirements, and TLS specificity during bypass of cyclobutane pyrimidine dimers (CPDs), abasic sites, and benzoa pyrene diol epoxide (BPDE) adducts. Our findings serve as basis for comparative analysis of the properties of proteins from different species and provided an important insight into the functional evolution of the Y-family polymerases. Apart from the expected increased thermostability of the T. lanuginosus Y-family pols, their major biochemical properties are very similar to properties of their human counterparts. In particular, both Rad30B homologs (T. lanuginosus and human pol) exhibit remarkably low fidelity during DNA synthesis that is template sequence dependent. It was previously hypothesized that higher organisms had acquired this property during eukaryotic evolution, but these observations imply that pol iota originated earlier than previously known, suggesting a critical cellular function in both lower and higher eukaryotes. Pathogenic bacteria pose a major global threat through the precipitous emergence of multidrug resistant strains (https://www.cdc.gov/drugresistance/biggest-threats.html). Horizontal transfer of mobile genetic elements including R-plasmids, integrative-conjugative elements (ICEs) and chromosomal instabilities accompanied by high mutation rates are among the key factors driving antibiotic resistance. Recent data have provided new insights into sources of mutagenesis leading to drug resistance. Elevated mutagenesis accompanying the induction of the SOS stress response caused by exposure to antibiotics and, more generally, to a wide variety of exogeneous DNA damage, have been linked to development of bacterial antibiotic resistance. For example, exposure of clinical isolates of E. coli to ciprofloxacin or zidovudine and Acinetobacter baumannii to UV or MMS resulted in the development of antibiotic resistance. There is a paucity of data documenting specific contributions of SOS-induced proteins, including pathogenic bacterial homologs of the E. coli LexA repressor, RecA, and low-fidelity DNA polymerase V (pol V), toward the acquisition of drug resistance. Homologs of pol V have been identified in a variety of Gammaproteobacteria, many of which are pathogenic. In addition to homologs encoded chromosomally, many pol V homologs are found on mobile genetic elements that can be horizontally transferred between different bacterial species. We referred to generic pol V homologs encoded on the chromosomes of pathogens by the term Pathogen Encoded Pols, abbreviated as PEPols. Homologous enzymes encoded by mobile elements, particularly integrative conjugative elements, or ICEs, were referred to as Mobile Element encoded Pols (MEPols). In collaboration with Myron Goodmans laboratory at the University of Southern California, we used Rum pol (RumA2B), from the integrative conjugative element (ICE), R391, as a model mobile element-encoded polymerase (MEPol). The highly mutagenic Rum pol is transferred horizontally into a variety of recipient cells, including many pathogens. Moving between species, it is unclear if Rum pol can function on its own or requires activation by host factors. To test this hypothesis, we investigated the biochemical and in vivo mutagenic behavior of Rum Mut assembled with RecA homologs purified from seven bacterial species; four clinical bacterial isolates in which the rumAB encoding R391/SXT family of ICEs have been previously identified (E. coli, V. cholerae, P. rettgeri, K. pneuomniae), and from three clinical isolates lacking rumAB genes (P. aeruginosa, M. tuberculosis, S. aureus). First, we demonstrated that Rum pol biochemical activity requires the formation of a physical mutasomal complex, Rum Mut, containing RumA2B-RecA-ATP, with RecA being donated by each recipient bacteria. Interestingly, Rum Mut specific activities in vitro and mutagenesis rates in vivo depended on the phylogenetic distance of host-cell RecA from E. coli RecA. We hypothesized that Rum pol, which is a highly conserved and effective mobile catalyst of rapid evolution, has the potential to generate a broad mutational landscape that could serve to ensure bacterial adaptation in antibiotic-rich environments leading to the establishment of antibiotic resistance.