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Multi-Step Control of Homologous Recombination Via Mec1/ATR Suppresses Chromosomal Rearrangements

ABSTRACT: The Mec1/ATR kinase is crucial for genome stability, yet the mechanism by which it prevents gross chromosomal rearrangements (GCRs) remains unknown. Here we find that in cells with deficient Mec1 signaling, GCRs accumulate due to the deregulation of multiple steps in homologous recombination (HR). Mec1 primarily suppresses GCRs through its role in activating the canonical checkpoint kinase Rad53, which ensures the proper control of DNA end resection. Upon loss of Rad53 signaling and resection control, Mec1 becomes hyperactivated and triggers a salvage pathway in which the Sgs1 helicase is recruited to sites of DNA lesions via the 911-Dpb11 scaffolds and phosphorylated by Mec1 to favor heteroduplex rejection and limit HR-driven GCR accumulation. Fusing an ssDNA recognition domain to Sgs1 bypasses the requirement of Mec1 signaling for GCR suppression and nearly eliminates D-loop formation, thus preventing non-allelic recombination events. We propose that Mec1 regulates multiple steps of HR to prevent GCRs while ensuring balanced HR usage when needed for promoting tolerance to replication stress.

SUBMITTER: Mr. Bokun Xie

PROVIDER: S-SCDT-10_1038-S44318-024-00139-9 | biostudies-other |

REPOSITORIES: biostudies-other

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Multi-Step Control of Homologous Recombination by Mec1/ATR Ensures Robust Suppression of Gross Chromosomal Rearrangements.

Project description:The Mec1/ATR kinase is crucial for genome stability, yet the mechanism by which it prevents gross chromosomal rearrangements (GCRs) remains unknown. Here we find that in cells with deficient Mec1 signaling, GCRs accumulate due to the deregulation of multiple steps in homologous recombination (HR). Mec1 primarily suppresses GCRs through its role in activating the canonical checkpoint kinase Rad53, which ensures the proper control of DNA end resection. Upon loss of Rad53 signaling and resection control, Mec1 becomes hyperactivated and triggers a salvage pathway in which the Sgs1 helicase is recruited to sites of DNA lesions via the 911-Dpb11 scaffolds to favor heteroduplex rejection and limit HR-driven GCR accumulation. Fusing an ssDNA recognition domain to Sgs1 bypasses the requirement of Mec1 signaling for GCR suppression and nearly eliminates D-loop formation, thus preventing non-allelic recombination events. We propose that Mec1 regulates multiple steps of HR to prevent GCRs while ensuring balanced HR usage when needed for promoting tolerance to replication stress.

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Project description:This SuperSeries is composed of the SubSeries listed below.

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Gene editing of colocalized NCF1 loci is associated with homologous recombination and chromosomal rearrangements - CAST-seq

Project description:Despite lingering genotoxicity concerns, CRISPR-based genome editing has progressively entered the gene therapy field. Here we report that interactions between nucleotide sequences that are localized on the same chromosome and exhibit a high degree of homology contribute substantially to post-editing chromosomal rearrangements. We successfully employed corrective editing strategies at the NCF1 gene, as well as at the colocalized pseudogenes, NCF1B and NCF1C, in a human cell line model of p47phox-deficient chronic granulomatous disease and in patient-derived human hematopoietic stem cells. Upon genetic manipulation, an improved droplet digital PCR-based method identified cells with altered copy numbers, spanning megabases from the edited loci. We attributed the high aberration frequency to the interaction between repetitive sequences and their predisposition to recombination events. Our findings emphasize the need for careful evaluation of the target-specific genomic context, such as the presence of homologous regions, whose genome instability can constitute a risk factor for chromosomal rearrangements upon genome editing.

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Coupling of Homologous Recombination and the Checkpoint by ATR.

Project description:ATR is a key regulator of cell-cycle checkpoints and homologous recombination (HR). Paradoxically, ATR inhibits CDKs during checkpoint responses, but CDK activity is required for efficient HR. Here, we show that ATR promotes HR after CDK-driven DNA end resection. ATR stimulates the BRCA1-PALB2 interaction after DNA damage and promotes PALB2 localization to DNA damage sites. ATR enhances BRCA1-PALB2 binding at least in part by inhibiting CDKs. The optimal interaction of BRCA1 and PALB2 requires phosphorylation of PALB2 at S59, an ATR site, and hypo-phosphorylation of S64, a CDK site. The PALB2-S59A/S64E mutant is defective for localization to DNA damage sites and HR, whereas the PALB2-S59E/S64A mutant partially bypasses ATR for its localization. Thus, HR is a biphasic process requiring both high-CDK and low-CDK periods. As exemplified by the regulation of PALB2 by ATR, ATR promotes HR by orchestrating a "CDK-to-ATR switch" post-resection, directly coupling the checkpoint to HR.

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A phosphorylation-and-ubiquitylation circuitry driving ATR activation and homologous recombination.

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Replisome instability, fork collapse, and gross chromosomal rearrangements arise synergistically from Mec1 kinase and RecQ helicase mutations.

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