Molecular Mechanisms of Y-Family Translesion Polymerase Activity in Bacillus subtilis

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Cells must efficiently and accurately replicate their genetic material, yet this process is challenged by the presence of unrepaired DNA damage on the template strand. In the DNA damage tolerance pathway translesion synthesis (TLS), specialized translesion polymerases replicate damaged DNA, promoting cell survival under stress. Most TLS polymerases are lower fidelity than replicative DNA polymerases, and thus their activity must be tightly regulated under normal growth conditions to maintain genome stability. Conversely, stress-induced mutagenesis by TLS polymerases can promote cell survival under certain conditions, such as by contributing to the development of antibiotic resistance in bacteria. The gram-negative bacterium E. coli has served as a model species for mechanistic studies of TLS polymerase regulation, but it is not known whether the same principles apply in other bacterial species, including the model gram-positive bacterium B. subtilis, which has two Y-family TLS polymerases, Pol Y1 and Pol Y2. By combining biochemical and microbiological experiments with live-cell single-molecule imaging, we will provide a comprehensive picture of the spatial organization, dynamics, and molecular coordination of the TLS polymerases Pol Y1 and Pol Y2 in B. subtilis. This study will reveal new insights into how DNA replication fidelity is maintained during normal growth and will broaden our understanding of TLS and DNA damage tolerance in bacterial species beyond E. coli. Aim 1: Determine how TLS polymerases respond to replication perturbations in B. subtilis In E. coli, TLS polymerases are excluded from the replication fork during normal cellular growth but selectively enriched in response to replication perturbations. It is not known, however, whether B. subtilis Pol Y1 and Pol Y2 are regulated in a similar manner. We will use live-cell single-molecule imaging to visualize fluorescently- labeled Pol Y1 and Pol Y2 molecules during normal replication and upon DNA damage, allowing us to determine if and how they respond to replication perturbations. Aim 2: Elucidate the role of the DnaN clamp in coordinating Pol Y1 and Pol Y2 activity The bacterial replication processivity factor, the DnaN sliding clamp, interacts with a wide range of proteins involved in DNA replication and repair through a common binding site. Pol Y1 and Pol Y2 contain clamp-binding motifs (CBMs), short peptide sequences that bind to DnaN. We will combine biochemical and microbiological assays with live-cell single-molecule imaging to elucidate the role of DnaN in regulating TLS in B. subtilis. Aim 3: Determine whether and how interactions with SSB play a role in TLS polymerase recruitment Bacterial single-stranded DNA-binding proteins (SSBs) act as a conserved binding platform for DNA replication and repair proteins. In E. coli, the TLS polymerase Pol IV is selectively enriched at stalled replication forks through its interaction with SSB. We will determine whether SSB plays a similar role in TLS polymerase regulation in B. subtilis by combining biochemical binding assays with live-cell single-molecule imaging.