The Mechanism of Replication Stalling and Recovery within Repetitive DNA

Abstract

Accurate chromosomal DNA replication is essential to maintain genomic stability. Genetic evidence suggests that certain repetitive sequences impair replication, yet the underlying mechanism is poorly defined. Replication could be directly inhibited by the DNA template or indirectly, for example by DNA-bound proteins. Here, we reconstituted replication of mono-, di- and trinucleotide repeats in vitro using eukaryotic replisomes assembled from purified proteins. We found that structure-prone repeats are sufficient to impair replication. Whilst template unwinding was unaffected, leading strand synthesis was inhibited, leading to fork uncoupling. Synthesis through hairpin-forming repeats relied on replisome-intrinsic mechanisms, whereas synthesis of quadruplex-forming repeats required an extrinsic accessory helicase. DNA-induced fork stalling was mechanistically similar to that induced by leading strand DNA lesions, highlighting structure-prone repeats as an important potential source of replication stress. Thus, we propose that our understanding of the cellular response to replication stress also applies to stalling induced by repetitive sequences.

Replication-induced DNA secondary structures drive fork uncoupling and breakage

“…Stalling induced by a single G4-or i-motif-forming sequence was persistent, although relatively weak when compared to other types of structure-prone sequences 45 .…”

“…Although Ctf4 is usually excluded from our reactions as it has minimal effect on budding yeast replication 48 , we have included Ctf4 in our experiments with Chl1, so we can infer that the lack of rescue by Chl1 is not due to its inability to be recruited to replication forks. Previous work has shown that Pif1 can rescue forks stalled at poly(dG)n and poly(dC)n sequences 45 . Consistent with this, Pif1 was also able to rescue forks stalled at both G4s and i-motifs (Fig.…”

“…Substrates for replication assays were constructed by inserting repetitive sequences into a 9.8kb parental plasmid (pGC542) containing a single synthetic yeast replication origin (ARS306) which has been previously described 45 . Repetitive sequences were cloned into the MCS 3kb downstream of the origin and expanded using a strategy that employs synthetic oligos and type IIS restriction enzymes 86 .…”

“…Reactions were quenched by adding EDTA to a final concentration of 100 mM. Pulse-chase experiments were carried out as previously described 45 . During the pulse, unlabelled deoxyribonucleotide concentrations were as follows: 30 μM dCTP, dTTP, dGTP and 2.5 μM dATP.…”

“…To carry out the chase, unlabelled dATP was added to a final concentration of 400 µM, with the addition of 400 µM of dGTP, dCTP or dTTP, or 800 µM dCTP where indicated. Repriming experiments were carried out by the addition of 60 nM oligonucleotide after loading and before initiation of replication 45 .…”