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. 2022 Jul 19;13(1):3953.
doi: 10.1038/s41467-022-31657-x.

The mechanism of replication stalling and recovery within repetitive DNA

Corella S Casas-Delucchi #  1 , Manuel Daza-Martin #  1 , Sophie L Williams  1 , Gideon Coster  2
Affiliations

The mechanism of replication stalling and recovery within repetitive DNA

Corella S Casas-Delucchi et al. Nat Commun. .

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 reconstitute replication of mono-, di- and trinucleotide repeats in vitro using eukaryotic replisomes assembled from purified proteins. We find that structure-prone repeats are sufficient to impair replication. Whilst template unwinding is unaffected, leading strand synthesis is inhibited, leading to fork uncoupling. Synthesis through hairpin-forming repeats is rescued by replisome-intrinsic mechanisms, whereas synthesis of quadruplex-forming repeats requires an extrinsic accessory helicase. DNA-induced fork stalling is 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 may also be applied to DNA-induced replication stalling.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. (CGG)n repeats induce orientation-dependent leading strand stalling.
A Schematic of the replication substrate used in this study. The ARS306 origin of replication drives sequence-specific initiation events. Two forks emanating from the origin generate a leftward moving 1.5 kb leading strand (“Left leading”) and a rightward moving 8.2 kb leading strand (“Right leading”). Various repeats were cloned 3 kb downstream such that only the rightward moving fork would encounter them. Thus, the left leading product serves as an internal control. Analysis of in vitro replication reactions by denaturing gel electrophoresis, using linear substrates with different types of leading strand repeats, in the presence (B) or absence (C) of pol δ. Replication reactions carried out in the absence of pol δ with a series of substrates containing increasing numbers of either (CGG)n (D) or (CCG)n (E) leading strand repeats, as well as a comparison to a site-specific leading strand CPD.
Fig. 2
Fig. 2. (CG)n, (G)n and (C)n also induce leading strand stalling.
A Replication reactions carried out in the absence of pol δ with a series of substrates containing increasing numbers of (CG)n, as well as a comparison to a site-specific leading strand CPD. Replication reactions carried out in the absence of pol δ, comparing two randomly generated scrambled sequences with the same base-pair composition and strand bias as either (CG)24 (B) or (CGG)21 (C). Replication reactions carried out in the absence of pol δ with a series of substrates containing increasing numbers of either guanine (D) or cytosine (E) leading strand homopolymers. F Replication reactions carried out in the absence of pol δ with a series of substrates containing different leading strand homopolymer templates as indicated.
Fig. 3
Fig. 3. Pol δ drives recovery from (CG)24 and (CGG)61 stalls, but not (G)50 or (C)50.
A Replication reactions carried out with the indicated templates in the absence or presence of pol δ. Pulse-chase experiments carried out with the (CGG)61 template in the absence (B) or presence (C) of pol δ. Reactions were initiated with radiolabelled dATP for 10 min, chased with excess ‘cold’ dATP and samples taken at the indicated time points. D, E Same as in B, C but with the (CG)24 template. F Pulse-chase experiments carried out with the indicated template. Reactions were initiated in the absence of pol δ to generate a pre-existing stall. After a 10 min pulse, pol δ was either added with the chase or not and samples taken at the indicated time points.
Fig. 4
Fig. 4. DNA-induced stalls trigger helicase-polymerase uncoupling.
Replication reactions carried out with the indicated templates in the presence of a primer that anneals 265 nt downstream of the repeats or a scrambled control primer in the absence (A) or presence (B) of pol δ. Replication reactions carried out with the indicated templates in the absence (C) or presence (D) of pol δ analysed by native gels. The insets show a longer exposure of the regions bound by the dashed boxes to better visualise uncoupled products.
Fig. 5
Fig. 5. Read-through of (CGG)n and (CG)n is facilitated by pol ε variants or elevated dNTPs.
A Replication reactions carried out with the indicated templates with different pol ε variants in the absence of pol δ. Quantification shown on the right is from five independent experiments. Shown is the mean, error bars are standard deviation. B Pulse-chase experiments carried out with the indicated templates in the absence (left panel) or presence (right panel) of pol δ. Reactions were initiated with radiolabelled dATP for 10 min and chased for another 10 min with either excess ‘cold’ dATP alone (dA) or with excess of all four dNTPs (dN).
Fig. 6
Fig. 6. Pif1 resolves DNA-induced stalls.
Pulse-chase experiments carried out with the indicated templates in the presence (A) or absence (B) of pol δ. Reactions were initiated with radiolabelled dATP. After a 10 min pulse, either WT or ATPase-dead (K264A) pif1 was added with the chase and samples taken after another 10 min.
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