Checkpoints are blind to replication restart and recombination intermediates that result in gross chromosomal rearrangements.

Saed Mohebi,Johanne M Murray,Antony M Carr,Ken’Ichi Mizuno,Adam Watson

doi:10.1038/ncomms7357

Saed Mohebi, Johanne M Murray + Show 3 more

Open Access

https://doi.org/10.1038/ncomms7357

Copy DOI

Journal: Nature Communications	Publication Date: Feb 27, 2015
Citations: 28	License type: CC BY 4.0

Affiliation: University of Sussex

Abstract

Replication fork inactivation can be overcome by homologous recombination, but this can cause gross chromosomal rearrangements that subsequently missegregate at mitosis, driving further chromosome instability. It is unclear when the chromosome rearrangements are generated and whether individual replication problems or the resulting recombination intermediates delay the cell cycle. Here we have investigated checkpoint activation during HR-dependent replication restart using a site-specific replication fork-arrest system. Analysis during a single cell cycle shows that HR-dependent replication intermediates arise in S phase, shortly after replication arrest, and are resolved into acentric and dicentric chromosomes in G2. Despite this, cells progress into mitosis without delay. Neither the DNA damage nor the intra-S phase checkpoints are activated in the first cell cycle, demonstrating that these checkpoints are blind to replication and recombination intermediates as well as to rearranged chromosomes. The dicentrics form anaphase bridges that subsequently break, inducing checkpoint activation in the second cell cycle.

Highlights

Replication fork inactivation can be overcome by homologous recombination, but this can cause gross chromosomal rearrangements that subsequently missegregate at mitosis, driving further chromosome instability
While this and several other lines of circumstantial evidence suggest that stochastic replication problems, NAHR-dependent recombination intermediates and the resulting chromosomal rearrangements do not generate robust checkpoint activation leading to mitotic delay, the stochastic nature of replication problems has precluded a definitive analysis of the role of DNA replication and DNA damage checkpoints in delaying mitosis in response to individual replication problems
By analysing site-specific replication restart in a single defined cell cycle we show that homologous recombination (HR)-dependent replication restart occurs in S phase, shortly after replication arrest, and that HR-dependent replication intermediates are resolved into acentric and capped chromosomes in G2

Summary

Introduction

Replication fork inactivation can be overcome by homologous recombination, but this can cause gross chromosomal rearrangements that subsequently missegregate at mitosis, driving further chromosome instability It is unclear when the chromosome rearrangements are generated and whether individual replication problems or the resulting recombination intermediates delay the cell cycle. Chromosome segregation defects are increased in Bloom’s syndrome cells but this has not been correlated with obvious mitotic delay[15,16] While this and several other lines of circumstantial evidence suggest that stochastic replication problems, NAHR-dependent recombination intermediates and the resulting chromosomal rearrangements do not generate robust checkpoint activation leading to mitotic delay, the stochastic nature of replication problems has precluded a definitive analysis of the role of DNA replication and DNA damage checkpoints in delaying mitosis in response to individual replication problems. It should be noted that replication of the capped chromosome would generate a dicentric

Methods

Results

Conclusion