Biology subjects

Dewar, J. M.

Publications and source records attributed to Dewar, J. M..

7 recordsLinked to original sources

bioRxiv2026

Topoisomerase IIIα resolves inter- and intra-molecular intertwines during DNA replication

Resolution of topological stress is crucial for genome integrity. Vertebrate topoisomerase II (TOP2) resolves catenanes to relieve topological stress and unlink daughter molecules during DNA replication. Topoisomerase III (TOP3) can also resolve catenanes, but its direct role during DNA replication, substrate specificity, and relevant binding partners remain unclear. Here we show that TOP3 becomes crucial when TOP2 function is compromised. We find that in Xenopus egg extracts, TOP3 promotes replication fork progression and daughter strand unlinking specifically during replication termination. TOP3 can carry out this role independently of its binding partners RMI1-RMI2 and the BLM helicase by acting on lagging-strand single-stranded DNA (ssDNA). Strikingly, elevated lagging-strand ssDNA drives formation of intramolecular ssDNA intertwines, which are ordinarily resolved by TOP3. Thus, TOP3 resolves intermolecular linkages to promote fork progression during termination and resolves intramolecular linkages that arise when high levels of ssDNA are present during DNA replication.

biochemistry↗

bioRxiv2026

Strand-independent degradation of uncoupled forks by EXO1 activates ATR and restrains synthesis

Nascent DNA degradation of replication forks shapes genome stability, yet the mechanism of degradation, and its direct consequences, remain unclear. Here we use localized nascent strand degradation at replication forks in Xenopus egg extracts to examine the mechanism and consequences of degradation. The exonuclease EXO1 is crucial for degradation and acts specifically on replication fork structures. Degradation requires EXO1 catalytic activity and is defective in the E109K Lynch Syndrome associated mutant. EXO1 degrades both nascent strands 5-3: the lagging strand from its native 5 end and the leading strand from a distal 5 entry on the sister fork, whereas leading strand 3 end remains stable. Impaired leading-strand degradation at a specific site does not affect degradation of the corresponding lagging-strand region, so the two strands are degraded independently. Degradation of the uncoupled fork has two downstream consequences. It is important to activate ATR, which is otherwise weakly activated by the uncoupled fork. Additionally, degradation restrains fork progression, independent of ATR activation. Our findings demonstrate that strand-independent degradation of uncoupled forks by EXO1 activates ATR and restrains fork progression

biochemistry↗

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↗

bioRxiv2022

RTEL1 and MCM10 overcome topological stress during vertebrate replication termination

Topological stress can cause replication forks to stall as they converge upon one another during termination of vertebrate DNA synthesis. However, replication forks ultimately overcome topological stress and complete DNA synthesis, suggesting that alternative mechanisms can overcome topological stress. We performed a proteomic analysis of converging replication forks that were stalled by topological stress in Xenopus egg extracts. We found that the helicase RTEL1 and the replisome protein MCM10 were highly enriched on DNA under these conditions. We show that RTEL1 normally plays a minor role during fork convergence while the role of MCM10 is normally negligible. However, RTEL1 and MCM10 both become crucially important for fork convergence under conditions of topological stress. RTEL1 and MCM10 exert non-additive effects on fork convergence and physically interact, suggesting that they function together. Furthermore, RTEL1 and MCM10 do not impact topoisomerase activity but do promote fork progression through a replication barrier. Thus, RTEL1 and MCM10 appear to play a general role in promoting progression of stalled forks, including when forks stall during termination. Overall, our data identify an alternate mechanism of termination involving RTEL1 and MCM10 that can be used to complete DNA synthesis under conditions of topological stress.

biochemistry↗

bioRxiv2021

Replication Fork Uncoupling Causes Nascent Strand Degradation and Fork Reversal

Genotoxins cause nascent strand degradation (NSD) and fork reversal during DNA replication. NSD and fork reversal are crucial for genome stability and exploited by chemotherapeutic approaches. However, it is unclear how NSD and fork reversal are triggered. Additionally, the fate of the replicative helicase during these processes is unknown. We developed a biochemical approach to study synchronous, localized NSD and fork reversal using Xenopus egg extracts. We show that replication fork uncoupling stimulates NSD of both nascent strands and progressive conversion of uncoupled forks to reversed forks. The replicative helicase remains bound during NSD and fork reversal, indicating that both processes take place behind the helicase. Unexpectedly, NSD occurs before and after fork reversal, indicating that multiple degradation steps take place. Overall, our data show that uncoupling causes NSD and fork reversal and identify key steps involved in these processes.

biochemistry↗

bioRxiv2021

Topoisomerase II poisons inhibit vertebrate DNA replication through distinct mechanisms

Topoisomerase II (Top2) unlinks chromosomes during vertebrate DNA replication. Top2 poisons are widely-used chemotherapeutics that stabilize Top2 complexes on DNA, leading to cytotoxic DNA breaks. However, it is unclear how these drugs affect DNA replication, which is a major target of Top2 poisons. Using Xenopus egg extracts, we show that the Top2 poisons etoposide and doxorubicin both inhibit DNA replication through different mechanisms. Etoposide induces Top2-dependent DNA breaks and induces Top2-dependent fork stalling by trapping Top2 behind replication forks. In contrast, doxorubicin does not lead to appreciable break formation and instead intercalates into parental DNA to inhibit replication fork progression. In human cells, etoposide stalls replication forks in a Top2-dependent manner, while doxorubicin stalls forks independently of Top2. However, both drugs exhibit Top2-dependent cytotoxicity. Thus, despite shared genetic requirements for cytotoxicity etoposide and doxorubicin inhibit DNA replication through distinct mechanisms.

biochemistry↗