Abstract
Bacillus subtilis PcrA interacts with the RNA polymerase and might contribute to mitigate replication–transcription conflicts (RTCs). We show that PcrA depletion lethality is partially suppressed by rnhB inactivation, but cell viability is significantly reduced by rnhC or dinG inactivation. Following PcrA depletion, cells lacking RnhC or DinG are extremely sensitive to DNA damage. Chromosome segregation is not further impaired by rnhB or dinG inactivation but is blocked by rnhC or recA inactivation upon PcrA depletion. Despite our efforts, we could not construct a ΔrnhC ΔrecA strain. These observations support the idea that PcrA dismantles RTCs. Purified PcrA, which binds single-stranded (ss) DNA over RNA, is a ssDNA-dependent ATPase and preferentially unwinds DNA in a 3′→5′direction. PcrA unwinds a 3′-tailed RNA of an RNA-DNA hybrid significantly faster than that of a DNA substrate. Our results suggest that a replicative stress, caused by mis-incorporated rNMPs, indirectly increases cell viability upon PcrA depletion. We propose that PcrA, in concert with RnhC or DinG, contributes to removing spontaneous or enzyme-driven R-loops, to counteract deleterious trafficking conflicts and preserve to genomic integrity.
Highlights
RNA polymerase (RNAP) transcription elongation complex is likely to encounter protein roadblocks and DNA lesions much more frequently than the replicative DNA polymerase (DNAP) moving along the same DNA template [1,2,3,4]
PcrA contributes to: (i) the removal of bulky lesions by nucleotide excision repair (NER), with UvrA acting as a DNA damage sensor and PcrA
RnhB), the endogenous threats generated by PcrA depletion were significantly reduced
Summary
RNA polymerase (RNAP) transcription elongation complex is likely to encounter protein roadblocks and DNA lesions much more frequently than the replicative DNA polymerase (DNAP) moving along the same DNA template [1,2,3,4]. These roadblocks cause a transient slowing-down or stalling of the replication forks, leading to a replication stress.
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