Abstract
PcrA depletion is lethal in wild-type Bacillus subtilis cells. The PcrA DNA helicase contributes to unwinding RNA from the template strand, backtracking the RNA polymerase, rescuing replication-transcription conflicts, and disassembling RecA from single-stranded DNA (ssDNA) by poorly understood mechanisms. We show that, in the presence of RecA, circa one PcrA/plasmid-size circular ssDNA (cssDNA) molecule hydrolyzes ATP at a rate similar to that on the isolated cssDNA. PcrA K37A, which poorly hydrolyses ATP, fails to displace RecA from cssDNA. SsbA inhibits and blocks the ATPase activities of PcrA and RecA, respectively. RecO partially antagonizes and counteracts the negative effect of SsbA on PcrA- and RecA-mediated ATP hydrolysis, respectively. Conversely, multiple PcrA molecules are required to inhibit RecA·ATP-mediated DNA strand exchange (DSE). RecO and SsbA poorly antagonize the PcrA inhibitory effect on RecA·ATP-mediated DSE. We propose that two separable PcrA functions exist: an iterative translocating PcrA monomer strips RecA from cssDNA to prevent unnecessary recombination with the mediators SsbA and RecO balancing such activity; and a PcrA cluster that disrupts DNA transactions, as RecA-mediated DSE.
Highlights
1A (SF1A) DNA helicases/translocases, which move in the 3′→5′ direction, are ubiquitous (Singleton et al, 2007; Lohman et al, 2008)
It has been shown that: 1) PcrA/UvrDEco contributes to nucleotide excision repair (NER) by displacing the excised damaged DNA segment; 2) PcrA/UvrDEco interacts with and induces RNA polymerase (RNAP) backtracking to alleviate replication-transcription conflict (RTC); 3) MfdEco, which interacts with the RNAP, contributes to transcription-coupled repair (TCR) and pushes the RNAP forward to correctly position its active site without interaction with the DNA; 4) inactivation of mfdEco suppresses the sensitivity to UV radiation in the ΔuvrDEco context; and 5) PcrA co-purifies with RecA as revealed by Tap-tag experiments
To understand the primary contribution of PcrA to NER, TCR, and RTCs or to limit unwanted recombination, the Δmfd pcrAT and ΔrecA pcrAT strains were exposed to the UV mimetic 4nitroquinoline 1-oxide (4NQO) and the survival rate analyzed under selective depletion of PcrA, with the recO16 pcrAT strain taken as a control in these experiments
Summary
1A (SF1A) DNA helicases/translocases, which move in the 3′→5′ direction, are ubiquitous (Singleton et al, 2007; Lohman et al, 2008) These enzymes, like those of the Proteobacteria [Rep (only present in γ-Proteobacteria) and UvrD], Actinobacteria (UvrD1 and UvrD2), Firmicutes (PcrA), and Ascomycota (Srs and Fbh1) phyla are crucial for DNA repair and repair-by-recombination (Singleton et al, 2007; Lohman et al, 2008; Epshtein, 2015).
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