Biology subjects

Cranford, M. T.

Publications and source records attributed to Cranford, M. T..

3 recordsLinked to original sources

bioRxiv2025

Resolution of collapsed forks is separate from completion of DNA synthesis

Replication fork collapse at single-strand DNA breaks (SSBs) poses a serious threat to genome stability. Using Xenopus egg extracts, we show that a replication fork encountering an SSB on either the leading- or lagging-strand template produces a single-ended double-strand break (seDSB). These broken ends are efficiently resolved by homologous recombination to yield D-loops and erroneous end-to-end fusions. Surprisingly, DNA synthesis downstream of an seDSB is highly inefficient. In contrast, when two forks converge at an SSB, they generate a double-ended DSB (deDSB) that efficiently completes DNA synthesis through double-strand break repair that is not dependent on homologous recombination. Leading, but not lagging, seDSBs can undergo extensive nucleolytic degradation that disassembles the divergent fork. These secondary collapse events efficiently resolve seDSBs but without completion of DNA synthesis. Moreover, PARP inhibition can enhance fork collapse at unmodified SSBs but not at abasic site SSBs, contrary to expectations. Our findings distinguish end resolution from replication completion and demonstrate flexibility in how PARP inhibition affects fork collapse.

biochemistry↗

bioRxiv2025

Leading and lagging strand abasic sites differentially affect vertebrate replisome progression but involve analogous bypass mechanisms

Abasic sites are one of the most frequent forms of DNA damage that interfere with DNA replication. However, abasic sites exhibit complex effects because they can be processed into other types of DNA damage. Thus, it remains poorly understood how abasic sites affect replisome progression, which replication-coupled repair pathways they elicit, and whether this is affected by the template strand that is damaged. Using Xenopus egg extracts, we developed an approach to analyze replication of DNA containing a site-specific, stable abasic site on the leading or lagging strand template. We show that abasic sites robustly stall synthesis of nascent DNA strands but exert different effects when encountered on the leading or lagging strand template. At a leading strand AP site, replisomes stall [~]100 bp from the lesion until it is bypassed or a converging fork triggers termination. At a lagging strand abasic site, replisome progression is unaffected and lagging strands are reprimed downstream, generating a post-replicative gap, which is then bypassed. Despite different effects on replisome progression, both leading and lagging strand abasic sites rely on translesion DNA synthesis for bypass. Our results detail similarities and differences between how leading and lagging strand AP sites affect vertebrate DNA replication.

biochemistry↗

bioRxiv2020

A hand-off of DNA between archaeal polymerases allows high-fidelity replication to resume at a discrete intermediate three bases past 8-oxoguanine

During DNA replication, the presence of 8-oxoguanine (8-oxoG) lesions in the template strand cause the high-fidelity (HiFi) DNA polymerase (Pol) to stall. An early response to 8-oxoG lesions involves on-the-fly translesion synthesis (TLS), in which a specialized TLS Pol is recruited and replaces the stalled HiFi Pol for bypass of the lesion. The length of TLS must be long enough for effective bypass, but it must also be regulated to minimize replication errors by the TLS Pol. The exact position where the TLS Pol ends and the HiFi Pol resumes (i.e. the length of the TLS patch) has not been described. We use steady-state and pre-steady-state kinetic assays to characterize lesion bypass intermediates formed by different archaeal polymerase holoenzyme complexes that include PCNA123 and RFC. After bypass of 8-oxoG by TLS PolY, products accumulate at the template position three base pairs beyond the lesion. PolY is catalytically poor for subsequent extension from this +3 position beyond 8-oxoG, but this inefficiency is overcome by rapid extension of HiFi PolB1. The reciprocation of Pol activities at this intermediate indicates a defined position where TLS Pol extension is limited and where the DNA substrate is handed back to the HiFi Pol after bypass of 8-oxoG.

biochemistry↗