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. polVR391 (Rum pol) DNA polymerase drives rapid bacterial drug resistance. In collaboration with Myron Goodman (University of Southern California), we investigated the acquisition of multidrug resistance by pathogenic bacteria, which is a well-documented existential threat for healthcare systems globally. Bacteria use two principal strategies to adapt to antibiotic stress. They can obtain antibiotic resistance genes through horizontal gene transfer of mobile genetic elements, and they can acquire mutations in genes involved in antibiotic uptake and metabolism. Mobile genetic elements, including SXT/R391 Integrative Conjugative Elements (ICEs), are widely spread among bacterial pathogens, including clinical isolates of Escherichia coli and numerous species of Vibrio, Shewanella and Klebsiella. SXT/R391 ICEs can facilitate rapid adaptation of host bacteria to changing environmental niches by contributing to genome plasticity and an expanded capacity to acquire novel traits, such as resistance to antibiotics, metals, and phage. SXT/R391 ICEs have been identified as key factors of antibiotic resistance in the seventh-pandemic lineage of V. cholerae, which remains a major cause of morbidity and mortality worldwide. Many ICEs encode homologs of the highly error-prone E. coli pol V (chromosomally encoded umuDC). Pol V (UmuD’2 C) is induced in E. coli as part of the SOS regulon and its expression is required to observe mutations above spontaneous background levels for cells irradiated with UV and exposed to chemicals that damage DNA. In R391, highly mutagenic polVR391 (Rum pol) is encoded by rumAB. Our collaborative study revealed that even under tight transcriptional and post-transcriptional regulation imposed by host bacteria and the R391 ICE itself, Rum pol rapidly accelerated development of Ciprofloxacin, Rifampicin and Ampicillin resistance in E. coli in response to SOS-inducing antibiotic and non-antibiotic external stressors Bleomycin, Ciprofloxacin and UV-radiation. The impact of Rum pol on the rate of acquisition of drug resistance appears to surpass potential contributions from other cellular processes. We also discovered that RecA protein plays a central role in controlling the ability of Rum pol to accelerate antibiotic resistance. Indeed, a single amino acid substitution in RecA, M197D, acts as a ‘Master Regulator’ that effectively eliminates the Rum pol-induced antibiotic resistance. We hypothesized that Rum pol should therefore be considered as one of the major factors driving development of de novo antibiotic resistance in pathogens carrying SXT/R391 ICEs.