Two mechanisms of chromosome fragility at replication-termination sites in bacteria

Abstract

Chromosomal fragile sites are implicated in promoting genome instability, which drives cancers and neurological diseases. Yet, the causes and mechanisms of chromosome fragility remain speculative. Here, we identify three spontaneous fragile sites in the Escherichia coli genome and define their DNA damage and repair intermediates at high resolution. We find that all three sites, all in the region of replication termination, display recurrent four-way DNA or Holliday junctions (HJs) and recurrent DNA breaks. Homology-directed double-strand break repair generates the recurrent HJs at all of these sites; however, distinct mechanisms of DNA breakage are implicated: replication fork collapse at natural replication barriers and, unexpectedly, frequent shearing of unsegregated sister chromosomes at cell division. We propose that mechanisms such as both of these may occur ubiquitously, including in humans, and may constitute some of the earliest events that underlie somatic cell mosaicism, cancers, and other diseases of genome instability.

Biology before the SOS Response—DNA Damage Mechanisms at Chromosome Fragile Sites

“…The spontaneous HJ foci also appear dependently on DNA replication, which suggests that the spontaneous ssDNA gaps result from replication. [16]. (DSB repair reviewed [33]).…”

“…We also learned that overproduction of the universal recombinase RecA provokes HJ-foci caused by replication-fork stalling and reversal, and that RecQ DNA helicase, the founding member of the conserved RecQ helicase family, prevents replication-fork reversal (Figure 1Biii) [18]. The promotion of reversed forks via RecA was observed in [16]. (DSB repair reviewed [33]).…”

“…The RecBCD-dependence of the HJ peaks rules out the possibility that they might be reversed replication forks (Figure 1Biii) as follows. (ii) Circular maps of spontaneous HJs (orange), obtained via X-seq [16,18], and DSB ends located using END-seq [38], in DSB repair-proficient wild-type (WT) and repair-deficient ∆recB cells [16]. DSBs at Ter sites are one-ended; DSBs surrounding dif, the site of chromosome decatenation, appear to be two-ended.…”

“…These sites are oriented such that replication forks that cross the terminus region are stopped, which prevents replication of the circular chromosome "backwards" towards the origin. Presumably, the Ter sites stall early-arriv- (ii) Circular maps of spontaneous HJs (orange), obtained via X-seq [16,18], and DSB ends located using END-seq [38], in DSB repair-proficient wild-type (WT) and repair-deficient ∆recB cells [16]. DSBs at Ter sites are one-ended; DSBs surrounding dif, the site of chromosome decatenation, appear to be two-ended.…”

“…The review that follows covers the mechanisms behind genome-destabilizing and human disease-instigating chromosome fragile sites, as inferred from the identification of fragile sites and mechanisms in E. coli [ 16 ]. The impression Miro made is likely to have influenced my lab’s: previous discovery of proteins of the human “DNA damageome” via an E. coli screen and identification of homologous proteins in humans that act similarly in human cells and cancers [ 17 ]; demonstration and use of a new role of E. coli RecQ DNA helicase to predict mechanistic features of human cancer evolution [ 18 ], also discussed here; efforts to create a universal toolbox of engineered proteins that capture DNA reaction intermediates in bacterial and human living cells, e.g., [ 18 , 19 ]; and work, over the whole of the lab’s operation, on stress-induced mutagenesis as a model for cancer and evolution across the tree of life [ 20 , 21 , 22 ], including bacterial antibiotic resistance [ 23 ].…”