MECHANISMS CONTROLLING THE INTEGRITY OF CONVERGING FORKS DURING REPLICATION TERMINATION

During S phase, natural fork pausing elements including replication termination zones (TERs) and transcribed genes, can easily lead to genotoxicity and chromosome fragility at fragile sites (CFs), known as hotspots for DNA breaks and chromosomal rearrangements, particularly in cancer cells. However, the key factors and pathways protecting the integrity of CFSs are not well understood. In order to elucidate these mechanisms, we used yeast as a model system, and combined genetic and genomic studies aimed at identifying fragile sites genome-wide. We found that centromeres, rDNA, telomeres and TERs represent hot spots for copy number variation (CNV) in wild-ype yeast cells exposed to replication stress. We found that top2 contributes to relieve the topological stress and extensive fork pausing during termination at TERs. We also observed that fragility at these sites is greatly enhanced in checkpoint mutants (Tel1-ATM1 and Mec1-ATR1), likely due to their inability to uncouple transcription from gene gating, thus leading to topological stress in front of replication forks. TER fragility in checkpoint defective cells can be partially rescued by Top2 and Condensin complex inactivation. We further demonstrated that Pif1 family helicase, Rrm3, regulate extensive fork pausing at TERs and endogenous pausing sites by suppressing RNA:DNA hybrid (R-loops) accumulation. Alongside, a RNA:DNA helicase Senataxin1 (Sen1) coordinates replication and transcription collision at TERs and endogenous fork pausing. Absence of Sen1 accumulates CNVs at TERs and double-strand DNA breaks. We show that programmed fork pausing and resolution of R-loops are key processes for TER integrity. Accordingly, sen1 rrm3 double mutants, accumulate gaps and RNA:DNA hybrids at TERs and fail to fuse replicons; moreover, these mutants exhibit unscheduled condensation events at TERs leading to chromosome entangling. Taken together, our data strongly suggests that, the replication checkpoint, Sen1 and Rrm3, coordinate replication termination to prevent accumulation of unsolved topological constrains and premature recruitment of Condensins and Top2.