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.

No abstract

The RecQ DNA helicase WRN is a synthetic lethal target for cancers with microsatellite instability (MSI), a form of genetic hypermutability arising from impaired mismatch repair 1-4 . WRN depletion induces widespread DNA double strand breaks (DSBs) in MSI cells, leading to cell cycle arrest and/or apoptosis. However, the mechanism by which WRN protects MSI cancers from DSBs remains unclear. Here, we demonstrate that TAdinucleotide repeats are highly unstable in MSI cells and exhibit surprisingly large-scale expansions, distinct from previously described insertion/deletion mutations of a few nucleotides 5 . We show that expanded TA repeats form non-B DNA secondary structures that stall replication forks, activate the ATR checkpoint kinase, and necessitate unwinding by the WRN helicase. In the absence of WRN, the expanded TA-dinucleotide repeats are susceptible to MUS81 nuclease cleavage, leading to massive chromosome shattering. Thus, our study uncovers a distinct biomarker within MSI tumors that underlies the synthetic lethal dependence on WRN, thereby supporting the development of WRN-based therapeutics.