부산대학교 도서관

DNA replication is essential for all living organisms. Several events can disrupt replication, including DNA damage (e.g., pyrimidine dimers, crosslinking) and so-called "roadblocks" (e.g., DNA-binding proteins or transcription). Bacteria have several well-characterized mechanisms for repairing damaged DNA and then restoring functional replication forks. However, little is known about the repair of stalled or arrested replication forks in the absence of chemical alterations to DNA. Using a library of random transposon insertions in Bacillus subtilis, we identified 35 genes that affect the ability of cells to survive exposure to an inhibitor that arrests replication elongation, but does not cause chemical alteration of the DNA. Genes identified include 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 chemical damage to DNA and loss of either nuclease causes increased sensitivity to DNA damaging agents. These 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 chemical damage to DNA, RecJ is detrimental to survival. 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 appeared to promote too much assembly of RecA filaments. Our results indicate that in the absence of chemical damage to DNA, RecA is dispensable for cells to survive replication arrest and that the stable RecA nucleofilaments favored by the RecJ pathway may lead to cell death by preventing proper processing of the arrested replication fork. Author summary: DNA replication is essential for life. A variety of events, including chemical damage to DNA (e.g., caused by irradiation with ultraviolet light) and other cellular stresses that do not cause chemical modification of the DNA, can perturb and stop ongoing replication. Cell survival often depends on the ability to resume (restart) replication after such disruptions and many organisms have multiple mechanisms for restarting replication. Many bacterial species have two pathways for restarting DNA replication following DNA damage (e.g., exposure to ultraviolet light), and loss of either of these pathways makes cells more sensitive to UV irradiation. We found that one of these two pathways is detrimental and the other pathway is beneficial for bacterial survival following replication arrest in the absence of chemical damage to DNA. These surprising findings highlight key differences between the two pathways and the fine balance organisms face in maintaining replication restart pathways that are sometimes helpful and other times harmful. [ABSTRACT FROM AUTHOR]