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Human BRCA2 protects the DNA when replication forks stall, whereas MRE11-RAD50 and WRN-DNA2 process or partially degrade these substrates. When mutated, these genes result in distinct genetic instabilities and cancers, arguing they have unique, not redundant, functions. Escherichia coli encodes functional homologs of MRE11-RAD50 (SbcC-SbcD), WRN-DNA2 (RecQ-RecJ), and BRCA2 (RecF). Here, we use 2-dimensional gels, pulse-labelling, and replication-profiling analysis to show the bacterial homologs act at distinct substrates and loci on the chromosome. Whereas RecF and RecJ-RecQ protect and process DNA at arrested replication forks to facilitate repair, RecBCD and SbcC-SbcD protect and process DNA at sites where forks converge. Comparing the assays used in E. coli to human cells, we consider whether these cellular roles may be functionally conserved.
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This content will become publicly available on September 8, 2026
Topo IV is required to allow replisomes to converge and complete replication on the chromosome
The ability to complete DNA replication as replisomes converge has recently been shown to be a highly regulated, multi-enzymatic process. Converging forks also are likely to generate unique supercoiled, tangled, or knotted substrates. These structures are typically resolved by one of the four topoisomerases encoded byEscherichia coli. However, identifying the cellular substrates and specific function for these essential enzymes which contain overlapping biochemical activities has remained challenging. Here, we show that Topo I and Topo IV are required to allow converging forks to complete chromosome replication. Impaired Topo I function leads to amplifications where forks converge, whereas inactivation of Topo IV prevents forks from converging and produces a dramatic loss of this chromosome region. The results are consistent with previous studies suggesting Topo I suppresses illegitimate initiations in the terminus region by disrupting R- and D-loops and demonstrate a specific requirement for Topo IV acting before replication completes to allow convergent forks to reach their doubling point. We propose that the positive supercoils arising between convergent forks are converted to precatenanes and resolved by Topo IV, when diminishing space may preclude gyrase from binding and functioning.
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- Award ID(s):
- 1916625
- PAR ID:
- 10642974
- Editor(s):
- Copenhaver, Gregory P
- Publisher / Repository:
- the Public Library of Science (PLOS)
- Date Published:
- Journal Name:
- PLOS Genetics
- Volume:
- 21
- Issue:
- 9
- ISSN:
- 1553-7404
- Page Range / eLocation ID:
- e1011857
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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