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UBC13-mediated template switching promotes replication stress resistance in FBH1-deficient cells.

ABSTRACT: The proper resolution of DNA damage during replication is essential for genome stability. FBH1, a UvrD, helicase plays crucial roles in the DNA damage response. FBH1 promotes double strand break formation and signaling in response to prolonged replication stress to initiate apoptosis. Human FBH1 regulates RAD51 to inhibit homologous recombination. A previous study suggested that mis-regulation of RAD51 may contribute to replication stress resistance in FBH1-deficient cells, but the underlying mechanism remains unknown. Here, we provide direct evidence that RAD51 promotes replication stress resistance in FBH1-deficient cells. We demonstrate inhibition of RAD51 using the small molecule, B02, partially rescues double strand break signaling in FBH1-deficient cells. We show that inhibition of only the strand exchange activity of RAD51 rescues double strand break signaling in FBH1 knockout cells. Finally, we show that depletion of UBC13, a E2 protein that promotes RAD51-dependent template switching, rescues double strand break formation and signaling sensitizing FBH1-deficient cells to replication stress. Our results suggest FBH1 regulates template switching to promote replication stress sensitivity.

SUBMITTER: Hawks AL

PROVIDER: S-EPMC10508767 | biostudies-literature | 2023 Sep

REPOSITORIES: biostudies-literature

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UBC13-mediated template switching promotes replication stress resistance in FBH1-deficient cells.

Hawks Alexandra L AL Bergmann Amy A McCraw Tyler J TJ Mason Jennifer M JM

bioRxiv : the preprint server for biology 20230919

The proper resolution of DNA damage during replication is essential for genome stability. FBH1, a UvrD, helicase plays crucial roles in the DNA damage response. FBH1 promotes double strand break formation and signaling in response to prolonged replication stress to initiate apoptosis. Human FBH1 regulates RAD51 to inhibit homologous recombination. A previous study suggested that mis-regulation of RAD51 may contribute to replication stress resistance in FBH1-deficient cells, but the underlying me ...[more]

PMID: 37732269

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Project description:Copy number variants (CNVs)-gains and losses of genomic sequences-are an important source of genetic variation underlying rapid adaptation and genome evolution. However, despite their central role in evolution little is known about the factors that contribute to the structure, size, formation rate, and fitness effects of adaptive CNVs. Local genomic sequences are likely to be an important determinant of these properties. Whereas it is known that point mutation rates vary with genomic location and local DNA sequence features, the role of genome architecture in the formation, selection, and the resulting evolutionary dynamics of CNVs is poorly understood. Previously, we have found that the GAP1 gene in Saccharomyces cerevisiae undergoes frequent and repeated amplification and selection under long-term experimental evolution in glutamine-limiting conditions. The GAP1 gene has a unique genomic architecture consisting of two flanking long terminal repeats (LTRs) and a proximate origin of DNA replication (autonomously replicating sequence, ARS), which are likely to promote rapid GAP1 CNV formation. To test the role of these genomic elements on CNV-mediated adaptive evolution, we performed experimental evolution in glutamine-limited chemostats using engineered strains lacking either the adjacent LTRs, ARS, or all elements. Using a CNV reporter system and neural network simulation-based inference (nnSBI) we quantified the formation rate and fitness effect of CNVs for each strain. We find that although GAP1 CNVs repeatedly form and sweep to high frequency in strains with modified genome architecture, removal of local DNA elements significantly impacts the rate and fitness effect of CNVs and the rate of adaptation. We performed genome sequence analysis to define the molecular mechanisms of CNV formation for 177 CNV lineages. We find that across all four strain backgrounds, between 26% and 80% of all GAP1 CNVs are mediated by Origin Dependent Inverted Repeat Amplification (ODIRA) which results from template switching between the leading and lagging strand during DNA synthesis. In the absence of the local ARS, a distal ARS can mediate CNV formation via ODIRA. In the absence of local LTRs, homologous recombination mechanisms still mediate gene amplification following de novo insertion of retrotransposon elements at the locus. Our study demonstrates the remarkable plasticity of the genome and reveals that template switching during DNA replication is a frequent source of adaptive CNVs.

Dataset Information

UBC13-mediated template switching promotes replication stress resistance in FBH1-deficient cells.

Publications

UBC13-mediated template switching promotes replication stress resistance in FBH1-deficient cells.

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