DNA replication stress causes chromosomal instability, especially at genomic regions termed fragile sites, the molecular basis of which is unclear. Tubbs et al. show that fragile-site instability is associated with replication-fork collapse and formation of DNA double-stranded breaks (DSBs) at poly(dA:dT) tracts, and that poly(dA:dT) tracts are also enriched and have a similar effect at strong origins of replication.

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To investigate replication-related DSB formation, the authors treated mouse and human cells with hydroxyurea, which causes replication elongation stress, and mapped DSB breakpoints genome-wide at nucleotide resolution. Many DSBs overlapped early-replicating fragile sites (ERFSs), and most of these DSBs overlapped replication initiation zones and formed in a replication-dependent manner, indicating that ERFSs are regions of fork collapse at active replication origins. DSBs in replication initiation zones were bordered by poly(dA) on the 3′ side and poly(dT) on the 5′ side, suggesting that poly(dA:dT) sequences near replication origins are hot spots of replication-fork stalling and collapse.

Following milder hydroxyurea treatment (and also in untreated cells), DSBs clustered also at AT-rich sequences at late-replicating common fragile sites (CFSs). The majority of these DSBs were within 20 bp of poly(dA:dT) tracts. Thus, replication-fork collapse at poly(dA:dT) tracts during replication stress probably contributes to chromosomal fragility at both ERFSs and CFSs.

The two ends (sides) of the DSBs were not symmetrical. Breaks at poly(dT) tracts reproducibly occurred at nucleotide position 10; indeed, the levels of DNA replication precipitously dropped at dT10 at left-moving replication forks and at dA10 at right-moving forks. Thus, forks under stress encountering a leading-strand template of poly(dT) stall around position T10 and produce well-defined breakpoints.

The breakpoints at the poly(dA) tracts occurred within long (250–400 nucleotide) stretches of unwound, single-stranded DNA (ssDNA), which is probably formed by the physical uncoupling of the unperturbed replication helicase and the stalled polymerase. In support of poly(dA) breakpoints occurring within ssDNA, the ssDNA binding protein replication protein A (RPA) was found to bind only the poly(dT) strand, thereby possibly exposing the poly(dA) tract to attack by nucleases and to DSB formation at random positions.

Replication origins in yeast are characterized by high AT content and nucleosome depletion, which could facilitate DNA unwinding and thus origin firing. By contrast, replication origins are poorly characterized in metazoans owing to their functional redundancy and lack of sequence consensus. The authors precisely mapped thousands of strong (frequently firing) replication origins in human and mouse cells, and found high AT content and considerable depletion of nucleosomes at the peaks of replication initiation zones. Thus, replication origin selection could be a conserved and regulated process.

Interestingly, similarly to fragile sites, sequences flanking these replication initiation peaks also exhibited asymmetrical nucleotide distribution, with higher poly(dA) content on the 3′ side and higher poly(dT) content on the 5′ side. Furthermore, hydroxyurea-induced DSBs were most frequent at poly(dA:dT) tracts located several hundred base pairs from the replication initiation peaks, raising the possibility that origin firing is naturally followed by ‘pausing’ at poly(dA:dT) tracts prior to the establishment of processive replication elongation.

forks under stress encountering a leading-strand template of poly(dT) stall … and produce well-defined breakpoints

In summary, ERFSs and CFSs share a common mechanism of fragility, which is based on fork stalling and collapse at poly(dA:dT) tracts. Such sequences also characterize strong replication origins, where they can cause fork collapse and DSBs. The role of poly(dA:dT) tracts in replication initiation could explain their high conservation and abundance in eukaryotic genomes, despite their inherent susceptibility to DSB formation.