Characterization of Unstudied Genes Important for Survival to DNA Damage and Damage-Independent Replication Fork Arrest
Creators: name: Wozniak, Katherine
Publisher: name: My University
Year: 2022
Subjects: subject: DNA damage and fork arrest
subject: genes of unknown function
subject: Molecular, Cellular and Developmental Biology
subject: Science

description: To ensure proper genetic inheritance, all bacteria must replicate and repair their DNA and coordinate each process with cell envelope synthesis for timely division. Bacteria are constantly exposed to exogenous and endogenous sources of DNA damage that can reduce cell fitness, increase mutation rates, or result in lethality if cells are unable to properly repair the damage and restart replication. In response, bacteria have evolved mechanisms for survival. Genes of unknown function represent greater than 40% of the total coding genes in Bacillus subtilis, even though B. subtilis is the most well studied Gram-positive organism to date. Genes of unknown function represent a major gap in our understanding of basic biological processes in B. subtilis and related organisms. Using high throughput genomics with drugs that inhibit DNA replication, we identified genes of unknown function for further study with the goal of improving our understanding of how B. subtilis responds to events that stalled DNA replication forks in vivo. Hydroxyurea (HU) is a bacteriostatic and bactericidal antineoplastic agent that inhibits ribonucleotide reductase responsible for conversion of rNTPs into dNTPs. Using Tn-seq following HU challenge, we identified genes important for mitigating DNA replication stress from dNTP depletion. We show that cell elongation occurs in response to increased formation of single stranded DNA with late activation of the SOS response after HU exposure. Using RNA-seq, we find that genes involved in dNTP synthesis are upregulated while genes coding for carbon uptake proteins are downregulated, which does not restore the dNTP balance and ends in activation of the SOS response after two hours. Using Illumina sequencing, we show that replication forks stall following HU treatment and that overexpression of ribonucleotide reductase reduces sensitivity to cells compromised for DNA repair. We show that B. subtilis upregulates transcription of catalases and peroxidases, as well as efflux pumps to detoxify HU as a mitigation strategy. Tn-seq with cells challenged with the DNA crosslinking agent MMC identified a putative kinase, ytmP, now named ccrZ for cell cycle regulator interacting with FtsZ. We show that cells with a ccrZ deletion are sensitive to a variety of DNA damaging agents. Further, cells lacking ccrZ are induced for the SOS response. Overexpression of ccrZ results in cell elongation and causes fork stalling near the chromosomal origin of replication similar to our finding with HU treatment. We show that CcrZ interacts with domains of the DNA replication initiation protein DnaA, as well as helicase loader DnaB. To understand the biochemical function of CcrZ we solved the crystal structure bound to AMPPNP and demonstrated ATPase activity in vitro on D-ribose and 2-deoxy-D-ribose. With these results, we conclude that CcrZ phosphorylates a second messenger to regulate DNA replication initiation and perhaps regulates initiation directly through protein interactions.