Biology subjects

Alani, E.

Publications and source records attributed to Alani, E..

8 recordsLinked to original sources

bioRxiv2025

The SIR2/3/4 complex promotes efficient break-induced replication by stabilizing recombination intermediates

Homologous recombination (HR) is one of the most effective pathways a cell can employ to repair DNA double strand breaks (DSBs). While most of the HR machinery has been characterized, less is known about how chromatin is reorganized and participates in repair. Here, we use a break-induced replication (BIR) assay, which measures conservative copying of a donor chromosome arm onto a single-ended recipient sequence, to explore a direct role for the SIR2/3/4 (SIR) silent chromatin complex in HR. Specifically, we measure an early step of BIR, D-loop extension, and simultaneously monitor localization of the SIR complex at the initiating DSB and an ectopic donor locus. Interestingly, we observe an [~]four- fold increase in Sir2 and Sir3 protein levels upon DSB induction and detect an enrichment of all three SIR complex subunits at the DSB and donor loci during periods of active D-loop extension. ChIP-seq experiments reveal enrichment of SIR complex subunits that extend up to 15 kb from the DSB site. This enrichment is substantially reduced in rad51{Delta} and rad52{Delta} mutants that show defects in BIR. Lastly, we show that sir mutations confer slowed D-loop extension kinetics between divergent substrates and increase the rate of mutation during BIR. These data are consistent with a model in which the SIR complex acts to stabilize the extending D-loop during BIR, leading to more efficient, and thus less mutagenic repair. Author SummaryDouble-strand breaks (DSBs) are among the most dangerous types of DNA lesions that occur in a cell; if unrepaired, they can cause cell death and deleterious chromosomal rearrangements. Homologous recombination (HR) is one of the most effective pathways to repair DSBs. While the machinery involved in HR has been characterized, less is known about how HR acts within a chromatin environment which contains DNA packaged into a compact state. In bakers yeast transcriptionally inactive "silent" chromatin is present at a limited number of domains in the cell. Bound to and regulating the transcriptional inactivity of these domains is the SIR complex. Curiously, various components of the SIR complex have been shown to localize to HR repaired DSBs. However, little is known about how they participate in HR. In this study we use a break-induced replication (BIR) assay, which measures conservative copying of a donor chromosome onto a single-ended recipient sequence, to characterize roles for the SIR complex in HR. We detect an enrichment of all three SIR complex subunits at the recipient and donor loci during periods of active repair. ChIP-seq, a method that detects protein-DNA interactions genome-wide, reveals enrichment of SIR complex subunits up to 15 kb from each side of the DSB site. This enrichment is significantly reduced in mutants that show defects in BIR. We assess the functional consequences of the loss of the SIR complex, showing that sir mutations slow early steps in BIR and increase the rate of mutation. Our data are consistent with a model in which the SIR complex acts to stabilize early intermediates in BIR, leading to more efficient, and thus less mutagenic repair.

molecular biology↗

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↗

bioRxiv2021

Exo1-protected DNA nicks direct crossover formation in meiosis

In most sexually reproducing organisms crossing over between chromosome homologs during meiosis is critical for the viability of haploid gametes. Most crossovers that form in meiosis in budding yeast result from the biased resolution of double Holliday Junction (dHJ) intermediates. This dHJ resolution step involves the actions Rad2/XPG family nuclease Exo1 and the Mlh1-Mlh3 mismatch repair endonuclease. At present little is known about how these factors act in meiosis at the molecular level. Here we show that Exo1 promotes meiotic crossing over by protecting DNA nicks from ligation. We found that structural elements in Exo1 required for interactions with DNA, such as bending of DNA during nick/flap recognition, are critical for its role in crossing over. Consistent with these observations, meiotic expression of the Rad2/XPG family member Rad27 partially rescued the crossover defect in exo1 null mutants, and meiotic overexpression of Cdc9 ligase specifically reduced the crossover levels of exo1 DNA binding mutants to levels approaching the exo1 null. In addition, our work identified a role for Exo1 in crossover interference that appears independent of its resection activity. Together, these studies provide experimental evidence for Exo1-protected nicks being critical for the formation of meiotic crossovers and their distribution.

molecular biology↗

bioRxiv2021

Handcuffing intrinsically disordered regions in Mlh1-Pms1 disrupts mismatch repair

The DNA mismatch repair (MMR) factor Mlh1-Pms1 contains long intrinsically disordered regions (IDRs). While essential for MMR, their exact functions remain elusive. We performed cross-linking mass spectrometry to identify the major interactions within the Mlh1-Pms1 heterodimer and used this information to insert FRB and FKBP dimerization domains into the IDRs of Mlh1 and Pms1. Yeast bearing these constructs were grown with rapamycin to induce dimerization. Strains containing FRB and FKBP domains in the Mlh1 IDR displayed complete MMR defects when grown with rapamycin, but removing rapamycin restored MMR functions. Furthermore, linking the Mlh1 and Pms1 IDRs through FRB-FKBP dimerization disrupted Mlh1-Pms1 binding to DNA, inappropriately activated Mlh1-Pms1, and caused MMR defects in vivo. We conclude that dynamic and coordinated rearrangements of the MLH IDRs regulate how the complex clamps DNA to catalyze MMR. The application of the FRB-FKBP dimerization system to interrogate in vivo functions of a critical repair complex will be useful for probing IDRs in diverse enzymes and to probe transient loss of MMR on demand.

genetics↗

bioRxiv2021

MLH1/3 variants causing aneuploidy, pregnancy loss, and premature reproductive aging

Most spontaneous pregnancy losses are a result of embryonic aneuploidy stemming from mis-segregation of chromosomes during meiosis. Proper disjunction of homologous chromosomes is dependent upon precise control of crossing-over, a process requiring the mismatch repair (MMR) genes MLH1 and MLH3. Both are required for fertility and completion of meiosis in mice. People inheriting variants in these genes are often at high risk for colorectal cancer and Lynch syndrome, yet the potential impacts of variants upon reproduction are unclear. To determine if MLH1/3 variants (namely single nucleotide polymorphisms, or SNPs) in human populations can cause reproductive abnormalities, we used a combination of computational predictions, yeast two-hybrid assays, and assays of MMR and recombination in yeast to select nine MLH1 and MLH3 variants for modeling in mice via genome editing. We identified 7 alleles that caused reproductive defects in mice including subfertility in females, male infertility, reduced sperm counts, and increased spermatocyte apoptosis. Remarkably, these alleles in females caused age-dependent decreases in litter size, and increased resorption of embryos during pregnancy. These outcomes were likely a consequence of reduced meiotic chiasmata, in turn causing an increase in misaligned chromosomes and univalents in meiotic metaphase I (MI). Our data indicate that segregating hypomorphic alleles of meiotic recombination genes in populations can predispose females to increased incidence of pregnancy loss from gamete aneuploidy.

genetics↗

bioRxiv2019

Mutation Of MLH3 Endonuclease Motif Reveals Integration Between Crossover Pathways In Mammalian Meiosis

During meiotic prophase I, double-strand breaks (DSBs) initiate homologous recombination leading to non-crossovers (NCOs) and crossovers (COs). In mouse, 10% of DSBs are designated to become COs, primarily through a pathway dependent on the MLH1-MLH3 heterodimer (MutL{gamma}). Mlh3 contains an endonuclease domain that is critical for resolving COs in yeast. We generated a mouse (Mlh3DN/DN) harboring a mutation within this conserved domain that is predicted to generate a protein that is catalytically inert. Mlh3DN/DN males, like fully null Mlh3-/- males, have no spermatozoa and are infertile, yet spermatocytes have normal DSBs and undergo normal synapsis events in early prophase I. Unlike Mlh3-/- males, mutation of the endonuclease domain within MLH3 permits normal loading and frequency of MutL{gamma} in pachynema. However, key DSB repair factors (RAD51) and mediators of CO pathway choice (BLM helicase) persist into pachynema in Mlh3DN/DN males, indicating a temporal delay in repair events and revealing a mechanism by which alternative DSB repair pathways may be selected. While Mlh3DN/DN spermatocytes retain only 22% of wildtype chiasmata counts, this frequency is greater than observed in Mlh3-/- males (10%), suggesting that the allele may permit partial endonuclease activity, or that other pathways can generate COs from these MutL{gamma}-defined repair intermediates in Mlh3DN/DN males. Double mutant mice homozygous for the Mlh3DN/DN and Mus81-/- mutations show losses in chiasmata that approach levels observed in Mlh3-/- males, indicating that the MUS81-EME1-regulated crossover pathway accounts for some of the increased residual chiasmata observed in Mlh3DN/DN spermatocytes. Our data demonstrate that mouse spermatocytes bearing the MLH1-MLH3DN/DN complex display the proper loading of factors essential for CO resolution (MutS{gamma}, CDK2, HEI10, MutL{gamma}). Despite these functions, mice bearing the Mlh3DN/DN allele show defects in the repair of meiotic recombination intermediates and a loss of most chiasmata. SUMMARYThe MLH1-MLH3 complex is essential for crossing over in mammalian meiosis. We generated a mutation in mouse MLH3 that alters its conserved endonuclease domain and show that it disrupts crossing over in a manner distinct from the full null Mlh3 mouse, but also results in male infertility.

genetics↗

bioRxiv2018

Intrinsically disordered regions regulate both catalytic and non-catalytic activities of the MutLα mismatch repair complex.

Intrinsically disordered regions (IDRs) are present in at least 30% of the eukaryotic proteome and are enriched in chromatin-associated proteins. Using a combination of genetics, biochemistry, and single-molecule biophysics, we characterize how IDRs regulate the functions of the yeast MutL (Mlh1-Pms1) mismatch repair (MMR) complex. Shortening or scrambling the IDRs in both subunits ablates MMR in vivo. Mlh1-Pms1 complexes with shorter IDRs that disrupt MMR retain wild-type DNA binding affinity but are impaired for diffusion on both naked and nucleosome-coated DNA. Moreover, the IDRs also regulate the ATP hydrolysis and nuclease activities that are encoded in the structured N- and C-terminal domains of the complex. This combination of phenotypes underlies the catastrophic MMR defect seen with the mutant MutL in vivo. More broadly, this work highlights an unanticipated multi-functional role for IDRs in regulating both facilitated diffusion on chromatin and nucleolytic processing of a DNA substrate.

biochemistry↗

bioRxiv2017

mlh3 separation of function and endonuclease defective mutants display an unexpected effect on meiotic recombination outcomes

Mlh1-Mlh3 is an endonuclease hypothesized to act in meiosis to resolve double Holliday junctions into crossovers. It also plays a minor role in eukaryotic DNA mismatch repair (MMR). To understand how Mlh1-Mlh3 functions in both meiosis and MMR, we analyzed in bakers yeast 60 new mlh3 alleles. Five alleles specifically disrupted MMR, whereas one (mlh3-32) specifically disrupted meiotic crossing over. Mlh1-mlh3 representatives for each separation of function class were purified and characterized. Both Mlh1-mlh3-32 (MMR+, crossover-) and Mlh1-mlh3-45 (MMR-, crossover+) displayed wild-type endonuclease activities in vitro. Msh2-Msh3, an MSH complex that acts with Mlh1-Mlh3 in MMR, stimulated the endonuclease activity of Mlh1-mlh3-32 but not Mlh1-mlh3-45, suggesting that Mlh1-mlh3-45 is defective in MSH interactions. Whole genome recombination maps were constructed for two mlh3 mutants with opposite separation of function phenotypes, and an endonuclease defective mutant. Unexpectedly, all three showed increases in the number of non-crossover events that were not observed in mlh3{Delta}. Our observations provide a structure-function map for Mlh3 that reveals the importance of protein-protein interactions in regulating Mlh1-Mlh3s enzymatic activity. They also illustrate how defective meiotic components can alter the fate of meiotic recombination intermediates, providing new insights for how meiotic recombination pathways are regulated.\n\nAuthor SummaryDuring meiosis, diploid germ cells that become eggs or sperm undergo a single round of DNA replication followed by two consecutive chromosomal divisions. The segregation of chromosomes at the first meiotic division is dependent in most organisms on at least one genetic exchange, or crossover event, between chromosome homologs. Homologs that do not receive a crossover frequently undergo non-disjunction at the first meiotic division, yielding gametes that lack chromosomes or contain additional copies. Such events have been linked to human disease and infertility. Recent studies suggest that the Mlh1-Mlh3 complex is an endonuclease that resolves recombination intermediates into crossovers. Interestingly, this complex also acts as a matchmaker in DNA mismatch repair (MMR) to remove DNA replication errors. How does one complex act in two different processes? We investigated this question by performing a mutational analysis of the bakers yeast Mlh3 protein. Five mutations were identified that disrupted MMR but not crossing over, and one mutation disrupted crossing over while maintaining MMR. Using a combination of biochemical and genetic analyses to further characterize these mutants we illustrate the importance of protein-protein interactions for Mlh1-Mlh3s activity. Importantly, we illustrate how defective meiotic components can alter the outcome of meiotic recombination events. They also provide new insights in our understanding of the basis of infertility syndromes.

genetics↗