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bioRxiv · 10.1101/2021.05.05.442863

Beneficial and detrimental genes in the cellular response to replication arrest

Schons-Fonseca, L.·Lazova, M. D.·Smith, J. L.·Grossman, A. D.
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Abstract

DNA replication is essential for all living organisms, and a variety of events can perturb replication, including DNA damage (e.g., pyrimidine dimers, crosslinking) and replication arrest due to "roadblocks" such as DNA-binding proteins or transcription. Bacteria have several well-characterized mechanisms for repairing damaged DNA and restoring functional replication forks. However, little is known about the repair of stalled or arrested replication forks in the absence of DNA lesions. Using a library of random transposon insertions in Bacillus subtilis, we identified 35 genes that affect the ability of cells to survive arrest of replication elongation, in the absence of DNA damage. Genes identified included those involved in iron-sulfur homeostasis, cell envelope biogenesis, and DNA repair and recombination. In B. subtilis, and many bacteria, two nucleases (AddAB and RecJ) are involved in early steps in repairing replication forks arrested by DNA damage. Loss of either one causes increased sensitivity to DNA damage. These single-strand nucleases resect DNA ends, leading to assembly of the recombinase RecA onto the single stranded DNA. Notably, we found that disruption of recJ increased survival of cells following replication arrest, indicating that, in the absence of DNA damage, RecJ is detrimental to surviving replication arrest. In contrast, and as expected, disruption of addA decreased survival of cells following replication arrest, indicating that AddA promotes survival. The different phenotypes of addA and recJ mutants appeared to be due to differences in assembly of RecA onto DNA. RecJ promoted too much assembly of RecA, and loss of RecA compensated for the detrimental effects of RecJ. Our results indicate that in the absence of DNA lesions, RecA is dispensable for cells to survive replication arrest and the stable RecA nucleofilaments favored by the RecJ pathway may lead to cell death by preventing proper processing of the arrested replication fork.

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Schons-Fonseca, L., Lazova, M. D., Smith, J. L., Grossman, A. D.. 2021-05-06. Beneficial and detrimental genes in the cellular response to replication arrest. https://doi.org/10.1101/2021.05.05.442863

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