The consequences of head-on replication-transcription conflicts on replication restart and genomic instability in Bacillus subtilis

Abstract

Concurrent bacterial replication and transcription lead to conflicts between the two machineries. These encounters, which impede replication and destabilize genomes, are especially detrimental when the replisome and RNA polymerase encounter each other head-on, on the lagging strand. Despite the negative consequences of conflicts, 26% of genes remain in the lagging strand orientation in Bacillus subtilis. My thesis research aimed to uncover the coping mechanisms activated and consequences experienced by cells in the event of head-on collisions between replication and transcription. The work herein first describes a mechanism required for replication restart in the immediate aftermath of collisions with transcription in B. subtilis. Subsequent chapters investigate the role of conflicts in shaping the B. subtilis genome over evolutionary time. We found that lagging strand genes experienced increased mutation rates compared to those on the leading strand, and experimentally demonstrated that transcription asymmetrically increases mutation rates for head-on genes. I then identified a cellular factor, the Y-family polymerase PolY1, that is required for asymmetric mutagenesis at conflict regions. PolY1 acts to promote mutagenesis at head-on genes through participation in transcription-coupled nucleotide excision repair, suggesting that conflicts may locally increase the susceptibility of the DNA to bulky lesions. Consistent with this model, I determined that transcription asymmetrically promotes pyrimidine dimer formation in head-on oriented genes. Overall the work presented in this dissertation provides new insight into mechanisms of genome maintenance and stability in B. subtilis, as well as the diverse effects of lagging strand transcription on DNA replication.