Person: Dewar, James M.
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Dewar
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James M.
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Dewar, James M.
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Publication FANCJ promotes DNA synthesis through G-quadruplex structures(BlackWell Publishing Ltd, 2014) Castillo Bosch, Pau; Segura-Bayona, Sandra; Koole, Wouter; van Heteren, Jane T; Dewar, James M.; Tijsterman, Marcel; Knipscheer, PuckOur genome contains many G-rich sequences, which have the propensity to fold into stable secondary DNA structures called G4 or G-quadruplex structures. These structures have been implicated in cellular processes such as gene regulation and telomere maintenance. However, G4 sequences are prone to mutations particularly upon replication stress or in the absence of specific helicases. To investigate how G-quadruplex structures are resolved during DNA replication, we developed a model system using ssDNA templates and Xenopus egg extracts that recapitulates eukaryotic G4 replication. Here, we show that G-quadruplex structures form a barrier for DNA replication. Nascent strand synthesis is blocked at one or two nucleotides from the G4. After transient stalling, G-quadruplexes are efficiently unwound and replicated. In contrast, depletion of the FANCJ/BRIP1 helicase causes persistent replication stalling at G-quadruplex structures, demonstrating a vital role for this helicase in resolving these structures. FANCJ performs this function independently of the classical Fanconi anemia pathway. These data provide evidence that the G4 sequence instability in FANCJ−/− cells and Fancj/dog1 deficient C. elegans is caused by replication stalling at G-quadruplexes.Publication DNA interstrand cross-link repair requires replication fork convergence(2014) Zhang, Jieqiong; Dewar, James M.; Budzowska, Magda; Motnenko, Anna; Cohn, Martin A.; Walter, JohannesDNA interstrand cross-links (ICLs) prevent strand separation during DNA replication and transcription and are therefore extremely cytotoxic. In metazoans, a major pathway of ICL repair is coupled to DNA replication and requires the Fanconi anemia pathway. In most current models, collision of a single DNA replication fork with an ICL is sufficient to initiate repair. In contrast, we show here that in Xenopus egg extracts, two DNA replication forks must converge on an ICL to trigger repair. When only one fork reaches the ICL, the replicative CMG helicase fails to unload from the stalled fork, and repair is blocked. Arrival of a second fork, even when substantially delayed, rescues repair. We conclude that ICL repair requires a replication-induced X-shaped DNA structure surrounding the lesion, and we speculate how this requirement helps maintain genomic stability in S phase.Publication The mechanism of DNA replication termination in vertebrates(2015) Dewar, James M.; Budzowska, Magda; Walter, JohannesEukaryotic DNA replication terminates when replisomes from adjacent replication origins converge. Termination involves local completion of DNA synthesis, decatenation of daughter molecules, and replisome disassembly. Termination has been difficult to study because termination events are generally asynchronous and sequence non-specific. To overcome these challenges, we paused converging replisomes with a site-specific barrier in Xenopus egg extracts. Upon removal of the barrier, forks underwent synchronous and site-specific termination, allowing mechanistic dissection of this process. We show that DNA synthesis does not slow detectably as forks approach each other and that leading strands pass each other unhindered before undergoing ligation to downstream lagging strands. Dissociation of CMG helicases occurs only after the final ligation step, and is not required for completion of DNA synthesis, strongly suggesting that converging CMGs pass one another and dissociate from double-stranded DNA. This termination mechanism allows rapid completion of DNA synthesis while avoiding premature replisome disassembly