Dual Roles of Poly(dA:dT) Tracts in Replication Initiation and Fork Collapse

Abstract

Replication origins, fragile sites, and rDNA have been implicated as sources of chromosomal instability. However, the defining genomic features of replication origins and fragile sites are among the least understood elements of eukaryote genomes. Here, we map sites of replication initiation and breakage in primary cells at high resolution. We find that replication initiates between transcribed genes within nucleosome-depleted structures established by long asymmetrical poly(dA:dT) tracts flanking the initiation site. Paradoxically, long (>20 bp) (dA:dT) tracts are also preferential sites of polar replication fork stalling and collapse within early-replicating fragile sites (ERFSs) and late-replicating common fragile sites (CFSs) and at the rDNA replication fork barrier. Poly(dA:dT) sequences are fragile because long single-strand poly(dA) stretches at the replication fork are unprotected by the replication protein A (RPA). We propose that the evolutionary expansion of poly(dA:dT) tracts in eukaryotic genomes promotes replication initiation, but at the cost of chromosome fragility.

A journey with common fragile sites: From S phase to telophase

“…Because they remain unprotected by the replication protein A (RPA), poly(dA) stretches would be prone to nuclease degradation, accounting for their instability. These features were observed both at ERFSs and CFSs, which lead the authors to propose that the two types of sites share the same mechanism of fragility . However, if the same mechanism operates at ERFSs and at CFSs, it remains to explain why, as discussed above, late replication is mandatory for CFS instability and why metaphase breaks map in the core of large transcribed fragile genes rather than in their 5′ and 3′ regions where initiation events also cluster.…”

“…These genomic regions include heterogeneous categories of loci named “fragile sites” that exhibit breaks on metaphase chromosomes, a phenomenon strongly accentuated upon replication stress . Presently, two main categories of fragile sites have been described in the genome of healthy individuals: common fragile sites (CFSs) and early replicating fragile sites (ERFSs) . Most CFSs nest in large genes replicating in mid to late S phase and display tissue‐dependent instability .…”

“…Most CFSs nest in large genes replicating in mid to late S phase and display tissue‐dependent instability . ERFSs frequently lie in inter‐genic sequences of early replicating regions that contain clusters of highly transcribed genes and, like CFSs, are tissue dependent . The interest in ERFSs and CFSs primarily stems from their key role in promoting DNA breaks and chromosome rearrangements associated with tumor progression .…”

The role of fork stalling and DNA structures in causing chromosome fragility

“…Mononucleotide runs, and especially A‐tracts, had the strongest correlation with cancer deletion breakpoints out of all potential non‐B‐DNA forming sequences probed in 46 000 cancer genomes . Interestingly, early replicating fragile sites are located close to origins and new data shows that poly(dA:dT) tracts adjacent to origins are preferential sites of fork stalling and fork collapse under conditions of HU‐induced replication stress in mouse B cells . DSB hotspots were also located by END‐seq at poly(dA:dT) tracts within the mouse WWOX gene in a region corresponding to the CFS FRA16D AT‐rich core in human cells and within the mouse FHIT gene which is the location of CFS FRA3B in humans; these data suggest that poly(dA:dT) tracts far from origins and within gene bodies are also fragile .…”

“…Interestingly, early replicating fragile sites are located close to origins and new data shows that poly(dA:dT) tracts adjacent to origins are preferential sites of fork stalling and fork collapse under conditions of HU‐induced replication stress in mouse B cells . DSB hotspots were also located by END‐seq at poly(dA:dT) tracts within the mouse WWOX gene in a region corresponding to the CFS FRA16D AT‐rich core in human cells and within the mouse FHIT gene which is the location of CFS FRA3B in humans; these data suggest that poly(dA:dT) tracts far from origins and within gene bodies are also fragile . The intergenic breaks at poly (dA:T) sites were present both when cells were exposed to a low dose of HU or no exogenous replication stress, emphasizing the role of sequence in their fragility.…”