Biology subjects

Can, G.

Publications and source records attributed to Can, G..

3 recordsLinked to original sources

bioRxiv2026

A cell-free system for mitotically-stimulated TMEJ

Double-stranded DNA breaks (DSBs) are cytotoxic and must be repaired to avoid cell death or mutagenesis. DSBs that form in the S or G2 phases of the cell cycle undergo end resection, creating substrates for homologous recombination (HR) and alternative end joining pathways. Theta-mediated end joining (TMEJ) is a DNA polymerase {theta} (Pol {theta}) dependent repair pathway that joins resected DSB ends by annealing short regions of microhomology. TMEJ becomes particularly important when HR is compromised or during mitosis. Despite intensive investigation, the mechanism of TMEJ remains poorly understood, in part due to the lack of an efficient cell-free system that recapitulates this reaction. Here, we address this deficit using Xenopus egg extracts. We show that a linear DNA template containing a 3' single-stranded DNA overhang with terminal microhomology undergoes efficient intermolecular joining in nucleoplasmic extracts derived from Xenopus eggs. Joining is abolished upon Pol {theta} depletion, and restoration of joining requires Pol {theta} that is proficient in both ATPase and DNA polymerase activities. Furthermore, joining is accelerated by recombinant Cyclin B-CDK1, which also enhances the recruitment of Pol {theta} to single-stranded DNA ends. This assay provides a biochemically tractable approach for dissecting the mechanism and regulation of TMEJ in a physiologically complex environment.

molecular biology↗

bioRxiv2024

TTF2 promotes replisome eviction from stalled forks in mitosis

Cells entering mitosis with incompletely replicated DNA face catastrophic chromosome segregation failure. During interphase, the replisome-associated E3 ubiquitin ligase TRAIP ubiquitylates barriers in front of the fork to allow replisome progression. In mitosis, TRAIP is reprogrammed from a trans-acting to a cis-acting ligase that can ubiquitylate the replisome itself. This enables the processing of unreplicated DNA by promoting replisome disassembly, fork breakage, and joining of the broken chromosome arms. Here, we describe a mechanism for this reprogramming: the ATPase TTF2 is recruited to the replisome, where its non-catalytic N-terminal domain tethers Cyclin B-CDK1-phosphorylated TRAIP to the leading strand DNA polymerase {varepsilon} in a geometry that allows replisome ubiquitylation. Thus, a phospho-regulated architectural switch alters replisome organization in mitosis to safeguard genome integrity before chromosome segregation.

biochemistry↗

bioRxiv2020

Checkpoint inhibition of origin firing prevents inappropriate replication outside of S-phase

Across eukaryotes, checkpoints maintain the order of cell cycle events in the face of DNA damage or incomplete replication. Although a wide array of DNA lesions activates the checkpoint kinases, whether and how this response differs in different phases of the cell cycle remains poorly understood. The S-phase checkpoint for example results in the slowing of replication, which in the budding yeast Saccharomyces cerevisiae is caused by Rad53 kinase-dependent inhibition of the initiation factors Sld3 and Dbf4. Despite this, we show here that Rad53 phosphorylates both of these substrates throughout the cell cycle at the same sites as in S-phase, suggesting roles for this pathway beyond S-phase. Indeed we show that Rad53-dependent inhibition of Sld3 and Dbf4 limits re-replication in G2/M phase, preventing inappropriate gene amplification events. In addition we show that inhibition of Sld3 and Dbf4 after DNA damage in G1 phase prevents premature replication initiation at all origins at the G1/S transition. This study redefines the scope and specificity of the S-phase checkpoint with implications for understanding the roles of this checkpoint in the majority of cancers that lack proper cell cycle controls.

cell biology↗