Mechanisms of Genome Maintenance and Mutagenesis in Bacillus subtilis.

Abstract

Deoxyribonucleic acid is used in all domains of life for the storage, replication and transmission of genetic information. Accurate replication of genomic DNA is essential to maintain gene function, and mechanisms have evolved to limit the mutagenic potential of DNA replication errors and impart stability to the genome. DNA replication and repair are well-studied, but there remain significant unanswered questions. In this dissertation I present my findings regarding the following questions: How does the mismatch repair protein MutS detect a single mismatch among millions of correctly paired nucleotides? Are there genome contexts that predispose loci to mutagenesis? What are the effects of unrepaired ribonucleotides in DNA on genome stability? Using a combination of biochemical, cell biological, genetic and genomic approaches, I have leveraged the power of the Bacillus subtilis model organism to show that mismatches produced during DNA replication are detected by MutS near their site of synthesis. This process is dependent on MutS interaction with replisome subunits. Using mutation accumulation lines, I have shown that local sequence contexts, and not global factors such as gene presence, direction of transcription and transcript abundance, affect replication error rate. I also reveal using mutation accumulation lines that ribonucleotides in bacterial DNA that go unrepaired cause GC to AT transitions. This is likely due to nucleotide excision repair effecting the removal of ribonucleotides in genomic DNA. This work provides important, novel insight to the fields of DNA mismatch repair and mutagenesis, and opens new avenues of exploration into the developing field of ribonucleotide excision repair.