Biology subjects

Crickard, J. B.

Publications and source records attributed to Crickard, J. B..

7 recordsLinked to original sources

bioRxiv2025

Rad54 separation of function mutation highlights unique roles during homologous recombination

Homologous recombination (HR) is a DNA repair pathway that utilizes a template-based approach to repair double-strand breaks within the genome. Template utilization requires the exchange of individual DNA strands, which members of the RecA family of recombinases facilitate. Rad51 is a primary strand exchange factor in eukaryotes. During regular mitotic DNA repair, Rad51 is aided by the DNA translocase Rad54, which acts as a motor to remodel the template DNA and stabilize primary-strand exchange intermediates. The regulation of this activity remains incompletely understood. Here, we have identified a conserved site within the C-terminal region of Rad54. The mutation of this site creates a functional separation at early strand-exchange intermediates in vivo. Using this mutant protein, we identify a novel intermediate essential for stabilizing displacement loop (D-loop) structures. This precedes the removal of Rad51 and DNA extension. Based on our experiments, we hypothesize that this Rad54 mutant cannot stabilize Rad51-mediated strand-exchange intermediates because it cannot topologically isolate dsDNA regions. Identifying a mutant that disrupts this intermediate before Rad51 removal unifies existing models of Rad54-mediated D-loop formation and extension.

biochemistry↗

bioRxiv2025

The Eukaryotic homology search complex distorts donor DNA structure to probe for homology

Homologous recombination (HR) is a DNA double-strand break repair pathway that facilitates genetic exchange and protects damaged replication forks during DNA synthesis. As a template-based repair process, the successful repair of a double-strand break depends on locating suitable homology from a donor DNA sequence elsewhere in the genome. In eukaryotes, Rad51 catalyzes the homology search in coordination with the ATP-dependent motor protein Rad54. The mechanism by which these two proteins regulate forces on dsDNA substrates during homology search remains unknown. Here, we have utilized single-molecule magnetic tweezers and optical trapping methods to monitor remodeling of the DNA template during the homology search. We find that the activity of Rad51 and Rad54 remodels the donor DNA substrate to control the association and dissociation of Rad51-ssDNA filaments in the absence of DNA homology. This mechanism occurs through the application of both linear (tension) and rotational (torsion) forces on the donor DNA. Finally, failure of Rad54 to act processively disrupts target selection in vivo. This study provides a basic understanding of how motorized homology search manipulates the donor DNA during the search for a suitable repair template. Significance StatementHomologous recombination (HR) is a double-strand DNA break repair pathway that utilizes a template-based target search process to locate a suitable homologous DNA sequence in the genome, thereby initiating DNA repair. Called the homology search, in eukaryotes, this process is carried out by the RecA family member Rad51. During the homology search, Rad51 collaborates with the motor protein Rad54 to identify and interrogate homologous DNA sequences within the genome. In this study, we have measured the forces applied by the combination of Rad51 and Rad54 to the donor DNA duplex. These measurements reveal a coordinated effort by these motor proteins to remodel donor DNA to probe for homology, shedding new light on how template-based homology searches interrogate the DNA strands.

biochemistry↗

bioRxiv2025

Evolution of Eukaryotic Specific DNA Binding Sites in Asgard Archaeal RecA Recombinases

Homologous recombination (HR) relies on the RecA-family recombinases Rad51 and Dmc1, which both catalyze DNA strand pairing and exchange but promote distinct recombination outcomes. How these recombinases became functionally specialized remains unclear. Here, we combine phylogenetic analysis with functional assays to define the basis of this specialization. We identify conserved amino acid variation in the DNA binding Loop 2 region across the archaeal eukaryotic lineage and show that these residues tune recombinase interactions with double-stranded DNA. Guided by these evolutionary signatures, we engineer Rad51 variants with altered DNA binding properties and test their functions. Rad51 like variants promote break-induced replication (BIR), whereas substitutions characteristic of Dmc1 suppress BIR and instead favor crossover outcomes, as measured using mitotic crossover assays. These results indicate that evolutionary divergence in Loop 2 alters recombinase binding on reaction intermediates, influencing their stability and progression toward distinct repair outcomes. Thus, evolutionary tuning of recombinase DNA interactions shift the balance between recombination pathways, providing a mechanistic basis for the functional specialization of Rad51 and Dmc1 during mitotic and meiotic HR.

evolutionary biology↗

bioRxiv2024

The translocation activity of Rad54 reduces crossover outcomes during homologous recombination

Homologous recombination (HR) is a template-based DNA double-strand break repair pathway that requires the selection of an appropriate DNA template for repair during the homology search stage of HR. Failure to execute the homology search quickly and efficiently can result in complex intermediates that generate genomic rearrangements, a hallmark of human cancers. Rad54 is an ATP dependent DNA motor protein that functions during the homology search by regulating the recombinase Rad51. How this regulation reduces genomic rearrangements is currently unknown. To better understand how Rad54 can prevent genomic rearrangements, we evaluated several amino acid mutations in Rad54 that were found in the COSMIC database. COSMIC is a collection of amino acid mutations identified in human cancers. These substitutions led to reduced Rad54 function and the discovery of a conserved motif in Rad54. Through genetic, biochemical, and single-molecule approaches, we show that disruption of this motif leads to failure in stabilizing early strand invasion intermediates, causing loss-of-heterozygosity rearrangements. Our study also suggests that the translocation rate of Rad54 is a determinant in balancing genetic exchange. This mechanism is likely fundamental to eukaryotic biology.

biochemistry↗

bioRxiv2023

The Dmc1 recombinase physically interacts with and promotes the meiotic crossoverfunctions of the Mlh1-Mlh3 endonuclease

The accurate segregation of homologous chromosomes during the Meiosis I reductional division in most sexually reproducing eukaryotes requires crossing over between homologs. In bakers yeast approximately 80 percent of meiotic crossovers result from Mlh1-Mlh3 and Exo1 acting to resolve double-Holliday junction (dHJ) intermediates in a biased manner. Little is known about how Mlh1-Mlh3 is recruited to recombination intermediates and whether it interacts with other meiotic factors prior to its role in crossover resolution. We performed a haploinsufficiency screen in bakers yeast to identify novel genetic interactors with Mlh1-Mlh3 using sensitized mlh3 alleles that disrupt the stability of the Mlh1-Mlh3 complex and confer defects in mismatch repair but do not disrupt meiotic crossing over. We identified several genetic interactions between MLH3 and DMC1, the recombinase responsible for recombination between homologous chromosomes during meiosis. We then showed that Mlh3 physically interacts with Dmc1 in vitro and at times in meiotic prophase when Dmc1 acts as a recombinase. Interestingly, restricting MLH3 expression to roughly the time of crossover resolution resulted in a mlh3 null-like phenotype for crossing over. Our data are consistent with a model in which Dmc1 nucleates a polymer of Mlh1-Mlh3 to promote crossing over.

genetics↗

bioRxiv2023

Rad53 phosphorylation of Rdh54 regulates the lifetime of homologous recombination intermediates

Rdh54 is a conserved DNA translocase that participates in homologous recombination (HR), DNA checkpoint adaptation, and chromosome segregation. Saccharomyces cerevisiae Rdh54 is a known target of the Mec1/Rad53 signaling axis, which globally protects genome integrity during DNA metabolism. While phosphorylation of DNA repair proteins by Mec1/Rad53 is critical for HR progression little is known about how specific post translational modifications alter HR reactions. Phosphorylation of Rdh54 is linked to protection of genomic integrity but the consequences of modification remain poorly understood. Here, we demonstrate that phosphorylation of the Rdh54 C-terminus by the effector kinase Rad53 regulates Rdh54 clustering activity as revealed by single molecule imaging. This stems from phosphorylation dependent and independent interactions between Rdh54 and Rad53. Genetic assays reveal that loss of phosphorylation leads to phenotypic changes resulting in loss-of-heterozygosity (LOH) outcomes. Our data highlight Rad53 as a key regulator of HR intermediates through activation and attenuation of Rdh54 motor function.

biochemistry↗

bioRxiv2022

Rdh54 stabilizes Rad51 at displacement loop intermediates to regulate genetic exchange between chromosomes

Homologous recombination (HR) is a double-strand break DNA repair pathway that preserves chromosome structure. To repair the damaged recipient DNA, HR requires an intact donor DNA sequence located elsewhere in the genome. After the double-strand break is repaired, DNA sequence information can be transferred between donor and recipient DNA molecules through different mechanisms, including DNA crossovers that form between homologous chromosomes. Regulating this transfer of information is an important step in effectively completing HR and maintaining genome integrity. For example, mitotic exchange of information between homologous chromosomes can result in loss-of-heterozygosity (LOH) in diploid organisms, and in higher eukaryotes, the development of cancer. The DNA motor protein Rdh54 is a highly conserved DNA translocase that functions during HR but has a limited role in repairing DNA. Instead, several existing phenotypes in rdh54{Delta} strains suggest that Rdh54 may regulate the flow of information between donor and recipient DNA molecules post DNA repair. In our current study, we used a combination of biochemical and genetic techniques to dissect the role of Rdh54 on the exchange of genetic information after DNA repair. Our data indicates that RDH54 regulates DNA sequence exchange between chromosomes by limiting the disruption of Rad51 at an early HR intermediate called the displacement loop (D-loop). Rdh54 also protects Rad51 filaments, acting in opposition to Rad51 removal by the DNA motor protein Rad54. Furthermore, we find that expression of a catalytically inactivate allele of Rdh54, rdh54K318R, displays a different distribution of information exchange outcomes than rdh54{Delta} cells. From these results, we propose a model for how Rdh54 may effectively regulate information transfer during homologous recombination.

biochemistry↗