Biology subjects

Maslowska, K.

Publications and source records attributed to Maslowska, K..

3 recordsLinked to original sources

bioRxiv2024

Rad51 determines pathway usage in post-replication repair

Stalled replication forks can be processed by several distinct mechanisms collectively called post-replication repair which includes homologous recombination, fork regression, and translesion DNA synthesis. However, the regulation of the usage between these pathways is not fully understood. The Rad51 protein plays a pivotal role in maintaining genomic stability through its roles in HR and in protecting stalled replication forks from degradation. We report the isolation of separation-of-function mutations in Saccharomyces cerevisiae Rad51 that retain their recombination function but display a defect in fork protection leading to a shift in post-replication repair pathway usage from HR to alternate pathways including mutagenic translesion synthesis. Rad51-E135D and Rad51-K305N show normal in vivo and in vitro recombination despite changes in their DNA binding profiles, in particular to dsDNA, with a resulting effect on their ATPase activities. The mutants lead to a defect in Rad51 recruitment to stalled forks in vivo as well as a defect in the protection of dsDNA from degradation by Dna2-Sgs1 and Exo1 in vitro. A high-resolution cryo-electron microscopy structure of the Rad51-ssDNA filament at 2.4 [A] resolution provides a structural basis for a mechanistic understanding of the mutant phenotypes. Together, the evidence suggests a model in which Rad51 binding to duplex DNA is critical to control pathway usage at stalled replication forks.

molecular biology↗

bioRxiv2021

Eukaryotic stress induced mutagenesis is limited by a local control of Translesion Synthesis

The DNA Damage Response (DDR) preserves the genetic integrity of the cell by sensing and repairing damages after a genotoxic stress. Translesion Synthesis (TLS), an error-prone DNA damage tolerance pathway, is controlled by PCNA ubiquitination. In this report, we raise the question whether TLS is controlled locally, or globally. Using a recently developed method that allows to follow the bypass of a single lesion inserted into the yeast genome, we show that: i) TLS is controlled locally at each individual lesion by PCNA ubiquitination, ii) a single lesion is enough to induce PCNA ubiquitination, and iii) PCNA ubiquitination is an imperative requirement for TLS to occur. More importantly, we show that global PCNA ubiquitination that follows a genotoxic stress does not increase TLS at individual lesions. We conclude that unlike the SOS response in bacteria, the eukaryotic DDR does not promote TLS and mutagenesis.

molecular biology↗

bioRxiv2019

iDamage: a method to integrate modified DNA into the yeast genome

In order to explore the mechanisms employed by living cells to deal with DNA alterations, we have developed a method by which we insert a modified DNA into a specific site of the yeast genome. This is achieved by the site-specific integration of a modified plasmid at a chosen locus of the yeast genome, through the use of the Cre/lox recombination system. In the present work, we have used our method to insert a single UV lesion into the yeast genome, and studied how the balance between error-free and error-prone lesion bypass is regulated. We show that the inhibition of homologous recombination, either directly (by the inactivation of rad51 recombinase) or through its control by preventing the poly-ubiquitination of PCNA (ubc13 mutant), leads to a strong increase in the use of TLS. Such regulatory aspects of the DNA damage tolerance could not have been observed with previous strategies using plasmid or randomly distributed lesions, which shows the advantage of our new method. The very robust and precise integration of any modified DNA at any chosen locus of the yeast genome that we describe here is a powerful tool that will allow exploration of many biological processes related to replication and repair of modified DNA.

molecular biology↗