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1.
Nucleic Acids Res ; 2024 Jun 03.
Artículo en Inglés | MEDLINE | ID: mdl-38828785

RESUMEN

Homologous recombination (HR) is a template-based DNA double-strand break repair pathway that requires the selection of an appropriate DNA sequence to facilitate repair. Selection occurs during a homology search that must be executed rapidly and with high fidelity. Failure to efficiently perform the homology search 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 exchanges is currently unknown. To better understand how Rad54 can reduce these outcomes, we evaluated several amino acid mutations in Rad54 that were identified 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 increased crossovers between homologous chromosomes. Our study also suggests that the translocation rate of Rad54 is a determinant in balancing genetic exchange. The latch domain's conservation implies an interaction likely fundamental to eukaryotic biology.

5.
Genetics ; 2024 Apr 24.
Artículo en Inglés | MEDLINE | ID: mdl-38657110

RESUMEN

The accurate segregation of homologous chromosomes during the Meiosis I reductional division in most sexually reproducing eukaryotes requires crossing over between homologs. In baker's 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 to perform its role in crossover resolution. We performed a gene dosage screen in baker's yeast to identify novel genetic interactors with Mlh1-Mlh3. Specifically, we looked for genes whose lowered dosage reduced meiotic crossing over 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. To our surprise we identified 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 in vivo. Partial complementation of Mlh3 crossover functions was observed when MLH3 was expressed under the control of the CLB1 promoter (NDT80 regulon), suggesting that Mlh3 function can be provided late in meiotic prophase at some functional cost. A model for how Dmc1 could facilitate Mlh1-Mlh3's role in crossover resolution is presented.

6.
Biochem Soc Trans ; 52(1): 367-377, 2024 Feb 28.
Artículo en Inglés | MEDLINE | ID: mdl-38323621

RESUMEN

Homologous recombination (HR) is a template-based DNA double-strand break repair pathway that functions to maintain genomic integrity. A vital component of the HR reaction is the identification of template DNA to be used during repair. This occurs through a mechanism known as the homology search. The homology search occurs in two steps: a collision step in which two pieces of DNA are forced to collide and a selection step that results in homologous pairing between matching DNA sequences. Selection of a homologous template is facilitated by recombinases of the RecA/Rad51 family of proteins in cooperation with helicases, translocases, and topoisomerases that determine the overall fidelity of the match. This menagerie of molecular machines acts to regulate critical intermediates during the homology search. These intermediates include recombinase filaments that probe for short stretches of homology and early strand invasion intermediates in the form of displacement loops (D-loops) that stabilize paired DNA. Here, we will discuss recent advances in understanding how these specific intermediates are regulated on the molecular level during the HR reaction. We will also discuss how the stability of these intermediates influences the ultimate outcomes of the HR reaction. Finally, we will discuss recent physiological models developed to explain how the homology search protects the genome.


Asunto(s)
ADN , Recombinación Homóloga , ADN/metabolismo , Reparación del ADN , ADN Helicasas/metabolismo , Roturas del ADN de Doble Cadena , Proteínas/genética
7.
Nucleic Acids Res ; 51(21): 11688-11705, 2023 Nov 27.
Artículo en Inglés | MEDLINE | ID: mdl-37850655

RESUMEN

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.


Asunto(s)
Recombinación Homóloga , Proteínas de Saccharomyces cerevisiae , Proteínas de Ciclo Celular/metabolismo , Quinasa de Punto de Control 2/genética , ADN/metabolismo , Daño del ADN , Fosforilación , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
8.
Methods Mol Biol ; 2599: 127-139, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-36427147

RESUMEN

Direct observation of enzymes that work to promote nucleic acid metabolism is a powerful approach to understanding their biochemical and biological properties. Over several years, fluorescent optical microscopy has developed as a powerful tool for watching biological pathways as they occur in real time. Here we describe DNA curtains as an optical microscopy tool that combines engineering, biochemistry, and single molecule imaging to make direct observations of enzymes as they work on DNA in real time. We will provide a detailed methodology of this approach including information about the setup of a basic TIRF microscope, assembly of flow chambers for imaging, and the protocol for making DNA curtains. Our goal is to help the reader better understand the technical approaches to DNA curtains and to better understand the biochemical and biological applications of this approach.


Asunto(s)
Membrana Dobles de Lípidos , Imagen Individual de Molécula , Membrana Dobles de Lípidos/química , Nanotecnología/métodos , ADN/química , Microscopía Fluorescente/métodos
9.
PLoS Genet ; 18(9): e1010412, 2022 09.
Artículo en Inglés | MEDLINE | ID: mdl-36099310

RESUMEN

Homologous recombination (HR) is a double-strand break DNA repair pathway that preserves chromosome structure. To repair damaged DNA, HR uses 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. Regulation of DNA sequence transfer 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), and in higher eukaryotes, the development of cancer. The DNA motor protein Rdh54 is a highly conserved DNA translocase that functions during HR. Several existing phenotypes in rdh54Δ strains suggest that Rdh54 may regulate effective exchange of DNA during HR. 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 during DNA repair. Our data indicate that RDH54 regulates DNA strand exchange by stabilizing Rad51 at an early HR intermediate called the displacement loop (D-loop). Rdh54 acts in opposition to Rad51 removal by the DNA motor protein Rad54. Furthermore, we find that expression of a catalytically inactivate allele of Rdh54, rdh54K318R, favors non-crossover outcomes. From these results, we propose a model for how Rdh54 may kinetically regulate strand exchange during homologous recombination.


Asunto(s)
Proteínas de Saccharomyces cerevisiae , Cromosomas/metabolismo , ADN/genética , ADN Helicasas/genética , Reparación del ADN/genética , ADN-Topoisomerasas/genética , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
10.
Proc Natl Acad Sci U S A ; 119(4)2022 01 25.
Artículo en Inglés | MEDLINE | ID: mdl-35042797

RESUMEN

Srs2 is a superfamily 1 (SF1) helicase that participates in several pathways necessary for the repair of damaged DNA. Srs2 regulates formation of early homologous recombination (HR) intermediates by actively removing the recombinase Rad51 from single-stranded DNA (ssDNA). It is not known whether and how Srs2 itself is down-regulated to allow for timely HR progression. Rad54 and Rdh54 are two closely related superfamily 2 (SF2) motor proteins that promote the formation of Rad51-dependent recombination intermediates. Rad54 and Rdh54 bind tightly to Rad51-ssDNA and act downstream of Srs2, suggesting that they may affect the ability of Srs2 to dismantle Rad51 filaments. Here, we used DNA curtains to determine whether Rad54 and Rdh54 alter the ability of Srs2 to disrupt Rad51 filaments. We show that Rad54 and Rdh54 act synergistically to greatly restrict the antirecombinase activity of Srs2. Our findings suggest that Srs2 may be accorded only a limited time window to act and that Rad54 and Rdh54 fulfill a role of prorecombinogenic licensing factors.


Asunto(s)
ADN Helicasas/metabolismo , Enzimas Reparadoras del ADN/metabolismo , ADN-Topoisomerasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/metabolismo , Daño del ADN/fisiología , ADN Helicasas/fisiología , Reparación del ADN/genética , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/fisiología , ADN-Topoisomerasas/fisiología , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Recombinación Homóloga/genética , Unión Proteica/genética , Recombinasa Rad51/metabolismo , Recombinasa Rad51/fisiología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiología
11.
EMBO J ; 39(20): e105705, 2020 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-32790929

RESUMEN

Rad54 and Rdh54 are closely related ATP-dependent motor proteins that participate in homologous recombination (HR). During HR, these enzymes functionally interact with the Rad51 presynaptic complex (PSC). Despite their importance, we know little about how they are organized within the PSC, or how their organization affects PSC function. Here, we use single-molecule optical microscopy and genetic analysis of chimeric protein constructs to evaluate the binding distributions of Rad54 and Rdh54 within the PSC. We find that Rad54 and Rdh54 have distinct binding sites within the PSC, which allow these proteins to act cooperatively as DNA sequences are aligned during homology search. Our data also reveal that Rad54 must bind to a specific location within the PSC, whereas Rdh54 retains its function in the repair of MMS-induced DNA damage even when recruited to the incorrect location. These findings support a model in which the relative binding sites of Rad54 and Rdh54 help to define their functions during mitotic HR.


Asunto(s)
Emparejamiento Cromosómico , ADN Helicasas/metabolismo , Enzimas Reparadoras del ADN/metabolismo , ADN-Topoisomerasas/metabolismo , ADN de Cadena Simple/metabolismo , Recombinasa Rad51/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sitios de Unión , Dominio Catalítico/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , ADN Helicasas/genética , Reparación del ADN/genética , Enzimas Reparadoras del ADN/genética , ADN-Topoisomerasas/genética , Proteínas de Unión al ADN/metabolismo , Mutación , Unión Proteica , Dominios Proteicos , Recombinasa Rad51/genética , Proteínas Recombinantes , Proteínas de Saccharomyces cerevisiae/genética
12.
Cell ; 181(6): 1380-1394.e18, 2020 06 11.
Artículo en Inglés | MEDLINE | ID: mdl-32502392

RESUMEN

Homologous recombination (HR) helps maintain genome integrity, and HR defects give rise to disease, especially cancer. During HR, damaged DNA must be aligned with an undamaged template through a process referred to as the homology search. Despite decades of study, key aspects of this search remain undefined. Here, we use single-molecule imaging to demonstrate that Rad54, a conserved Snf2-like protein found in all eukaryotes, switches the search from the diffusion-based pathways characteristic of the basal HR machinery to an active process in which DNA sequences are aligned via an ATP-dependent molecular motor-driven mechanism. We further demonstrate that Rad54 disrupts the donor template strands, enabling the search to take place within a migrating DNA bubble-like structure that is bound by replication protein A (RPA). Our results reveal that Rad54, working together with RPA, fundamentally alters how DNA sequences are aligned during HR.


Asunto(s)
Adenosina Trifosfato/genética , ADN Helicasas/genética , Enzimas Reparadoras del ADN/genética , ADN/genética , Recombinación Homóloga/genética , Proteínas de Saccharomyces cerevisiae/genética , Adenosina Trifosfatasas/genética , Daño del ADN/genética , Reparación del ADN/genética , Proteínas de Unión al ADN/genética , Hidrólisis , Saccharomyces cerevisiae/genética , Alineación de Secuencia/métodos
13.
Mol Cell ; 76(5): 699-711.e6, 2019 12 05.
Artículo en Inglés | MEDLINE | ID: mdl-31542296

RESUMEN

Rad52 is a key factor for homologous recombination (HR) in yeast. Rad52 helps assemble Rad51-ssDNA nucleoprotein filaments that catalyze DNA strand exchange, and it mediates single-strand DNA annealing. We find that Rad52 has an even earlier function in HR in restricting DNA double-stranded break ends resection that generates 3' single-stranded DNA (ssDNA) tails. In fission yeast, Exo1 is the primary resection nuclease, with the helicase Rqh1 playing a minor role. We demonstrate that the choice of two extensive resection pathways is regulated by Rad52. In rad52 cells, the resection rate increases from ∼3-5 kb/h up to ∼10-20 kb/h in an Rqh1-dependent manner, while Exo1 becomes dispensable. Budding yeast Rad52 similarly inhibits Sgs1-dependent resection. Single-molecule analysis with purified budding yeast proteins shows that Rad52 competes with Sgs1 for DNA end binding and inhibits Sgs1 translocation along DNA. These results identify a role for Rad52 in limiting ssDNA generated by end resection.


Asunto(s)
Roturas del ADN de Doble Cadena , Roturas del ADN de Cadena Simple , Reparación del ADN , ADN de Hongos/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimología , ADN Helicasas/genética , ADN Helicasas/metabolismo , ADN de Hongos/genética , Proteínas de Unión al ADN/genética , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Regulación Fúngica de la Expresión Génica , Cinética , Mutación , Dominios Proteicos , Transporte de Proteínas , Proteína Recombinante y Reparadora de ADN Rad52/genética , RecQ Helicasas/genética , RecQ Helicasas/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética
14.
Genes Dev ; 33(17-18): 1191-1207, 2019 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-31371435

RESUMEN

The vast majority of eukaryotes possess two DNA recombinases: Rad51, which is ubiquitously expressed, and Dmc1, which is meiosis-specific. The evolutionary origins of this two-recombinase system remain poorly understood. Interestingly, Dmc1 can stabilize mismatch-containing base triplets, whereas Rad51 cannot. Here, we demonstrate that this difference can be attributed to three amino acids conserved only within the Dmc1 lineage of the Rad51/RecA family. Chimeric Rad51 mutants harboring Dmc1-specific amino acids gain the ability to stabilize heteroduplex DNA joints with mismatch-containing base triplets, whereas Dmc1 mutants with Rad51-specific amino acids lose this ability. Remarkably, RAD-51 from Caenorhabditis elegans, an organism without Dmc1, has acquired "Dmc1-like" amino acids. Chimeric C. elegans RAD-51 harboring "canonical" Rad51 amino acids gives rise to toxic recombination intermediates, which must be actively dismantled to permit normal meiotic progression. We propose that Dmc1 lineage-specific amino acids involved in the stabilization of heteroduplex DNA joints with mismatch-containing base triplets may contribute to normal meiotic recombination.


Asunto(s)
Aminoácidos/metabolismo , Recombinasa Rad51/química , Recombinasa Rad51/metabolismo , Recombinasas/química , Recombinasas/metabolismo , Recombinación Genética/genética , Aminoácidos/genética , Animales , Disparidad de Par Base , Caenorhabditis elegans/enzimología , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Secuencia Conservada , Mutación , Recombinasa Rad51/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Recombinasas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
15.
Annu Rev Biophys ; 48: 255-273, 2019 05 06.
Artículo en Inglés | MEDLINE | ID: mdl-30857400

RESUMEN

Helicases are enzymes that move, manage, and manipulate nucleic acids. They can be subdivided into six super families and are required for all aspects of nucleic acid metabolism. In general, all helicases function by converting the chemical energy stored in the bond between the gamma and beta phosphates of adenosine triphosphate into mechanical work, which results in the unidirectional movement of the helicase protein along one strand of a nucleic acid. The results of this translocation activity can range from separation of strands within duplex nucleic acids to the physical remodeling or removal of nucleoprotein complexes. In this review, we focus on describing key helicases from the model organism Saccharomyces cerevisiae that contribute to the regulation of homologous recombination, which is an essential DNA repair pathway for fixing damaged chromosomes.


Asunto(s)
ADN Helicasas/metabolismo , Saccharomyces cerevisiae/enzimología , ADN Helicasas/genética , Genoma Fúngico , Recombinación Homóloga , Humanos , Saccharomyces cerevisiae/genética
16.
Proc Natl Acad Sci U S A ; 116(13): 6091-6100, 2019 03 26.
Artículo en Inglés | MEDLINE | ID: mdl-30850524

RESUMEN

In the repair of DNA double-strand breaks by homologous recombination, the DNA break ends must first be processed into 3' single-strand DNA overhangs. In budding yeast, end processing requires the helicase Sgs1 (BLM in humans), the nuclease/helicase Dna2, Top3-Rmi1, and replication protein A (RPA). Here, we use single-molecule imaging to visualize Sgs1-dependent end processing in real-time. We show that Sgs1 is recruited to DNA ends through Top3-Rmi1-dependent or -independent means, and in both cases Sgs1 is maintained in an immoble state at the DNA ends. Importantly, the addition of Dna2 triggers processive Sgs1 translocation, but DNA resection only occurs when RPA is also present. We also demonstrate that the Sgs1-Dna2-Top3-Rmi1-RPA ensemble can efficiently disrupt nucleosomes, and that Sgs1 itself possesses nucleosome remodeling activity. Together, these results shed light on the regulatory interplay among conserved protein factors that mediate the nucleolytic processing of DNA ends in preparation for homologous recombination-mediated chromosome damage repair.


Asunto(s)
Roturas del ADN de Doble Cadena , ADN Helicasas/metabolismo , Reparación del ADN , RecQ Helicasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Recombinación Homóloga , Nucleosomas/metabolismo , Proteína de Replicación A/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Imagen Individual de Molécula/métodos
17.
Nucleic Acids Res ; 47(9): 4694-4706, 2019 05 21.
Artículo en Inglés | MEDLINE | ID: mdl-30916344

RESUMEN

DNA helicases of the RecQ family are conserved among the three domains of life and play essential roles in genome maintenance. Mutations in several human RecQ helicases lead to diseases that are marked by cancer predisposition. The Saccharomyces cerevisiae RecQ helicase Sgs1 is orthologous to human BLM, defects in which cause the cancer-prone Bloom's Syndrome. Here, we use single-molecule imaging to provide a quantitative mechanistic understanding of Sgs1 activities on single stranded DNA (ssDNA), which is a central intermediate in all aspects of DNA metabolism. We show that Sgs1 acts upon ssDNA bound by either replication protein A (RPA) or the recombinase Rad51. Surprisingly, we find that Sgs1 utilizes a novel motor mechanism for disrupting ssDNA intermediates bound by the recombinase protein Rad51. The ability of Sgs1 to disrupt Rad51-ssDNA filaments may explain some of the defects engendered by RECQ helicase deficiencies in human cells.


Asunto(s)
Recombinasa Rad51/genética , RecQ Helicasas/genética , Recombinación Genética , Proteínas de Saccharomyces cerevisiae/genética , Adenosina Trifosfato/genética , Síndrome de Bloom/genética , Síndrome de Bloom/patología , Reparación del ADN/genética , ADN de Cadena Simple , Humanos , Mutación/genética , Saccharomyces cerevisiae/genética
18.
Methods ; 159-160: 51-58, 2019 04 15.
Artículo en Inglés | MEDLINE | ID: mdl-30660864

RESUMEN

Transcription elongation through the nucleosome is a precisely coordinated activity to ensure timely production of RNA and accurate regulation of co-transcriptional histone modifications. Nucleosomes actively participate in transcription regulation at various levels and impose physical barriers to RNA polymerase II (RNAPII) during transcription elongation. Despite its high significance, the detailed dynamics of how RNAPII translocates along nucleosomal DNA during transcription elongation and how the nucleosome structure dynamically conforms to the changes necessary for RNAPII progression remain poorly understood. Transcription elongation through the nucleosome is a complex process and investigating the changes of the nucleosome structure during this process by ensemble measurements is daunting. This is because it is nearly impossible to synchronize elongation complexes within a nucleosome or a sub-nucleosome to a designated location at a high enough efficiency for desired sample homogeneity. Here we review our recently developed single-molecule FRET experimental system and method that has fulfilled this deficiency. With our method, one can follow the changes in the structure of individual nucleosomes during transcription elongation. We demonstrated that this method enables the detailed measurements of the kinetics of transcription elongation through the nucleosome and its regulation by a transcription factor, which can be easily extended to investigations of the roles of environmental variables and histone post-translational modifications in regulating transcription elongation.


Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Nucleosomas/metabolismo , ARN Polimerasa II/metabolismo , Elongación de la Transcripción Genética , Factores de Transcripción/metabolismo , Eucariontes/enzimología , Eucariontes/genética , Eucariontes/metabolismo , Cinética , Imagen Individual de Molécula/métodos , Levaduras/enzimología , Levaduras/genética , Levaduras/metabolismo
19.
J Biol Chem ; 294(2): 490-501, 2019 01 11.
Artículo en Inglés | MEDLINE | ID: mdl-30420424

RESUMEN

Homologous recombination (HR) is a universally conserved DNA repair pathway that can result in the exchange of genetic material. In eukaryotes, HR has evolved into an essential step in meiosis. During meiosis many eukaryotes utilize a two-recombinase pathway. This system consists of Rad51 and the meiosis-specific recombinase Dmc1. Both recombinases have distinct activities during meiotic HR, despite being highly similar in sequence and having closely related biochemical activities, raising the question of how these two proteins can perform separate functions. A likely explanation for their differential regulation involves the meiosis-specific recombination proteins Hop2 and Mnd1, which are part of a highly conserved eukaryotic protein complex that participates in HR, albeit through poorly understood mechanisms. To better understand how Hop2-Mnd1 functions during HR, here we used DNA curtains in conjunction with single-molecule imaging to measure and quantify the binding of the Hop2-Mnd1 complex from Saccharomyces cerevisiae to recombination intermediates comprising Rad51- and Dmc1-ssDNA in real time. We found that yeast Hop2-Mnd1 bound rapidly to Dmc1-ssDNA filaments with high affinity and remained bound for ∼1.3 min before dissociating. We also observed that this binding interaction was highly specific for Dmc1 and found no evidence for an association of Hop2-Mnd1 with Rad51-ssDNA or RPA-ssDNA. Our findings provide new quantitative insights into the binding dynamics of Hop2-Mnd1 with the meiotic presynaptic complex. On the basis of these findings, we propose a model in which recombinase specificities for meiotic accessory proteins enhance separation of the recombinases' functions during meiotic HR.


Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Mapas de Interacción de Proteínas , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/análisis , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/análisis , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/metabolismo , Recombinación Homóloga , Meiosis , Unión Proteica , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/análisis
20.
Cell Cycle ; 17(23): 2520-2530, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30482074

RESUMEN

Meiosis is the basis for sexual reproduction and is marked by the sequential reduction of chromosome number during successive cell cycles, resulting in four haploid gametes. A central component of the meiotic program is the formation and repair of programmed double strand breaks. Recombination-driven repair of these meiotic breaks differs from recombination during mitosis in that meiotic breaks are preferentially repaired using the homologous chromosomes in a process known as homolog bias. Homolog bias allows for physical interactions between homologous chromosomes that are required for proper chromosome segregation, and the formation of crossover products ensuring genetic diversity in progeny. An important aspect of meiosis in the differential regulation of the two eukaryotic RecA homologs, Rad51 and Dmc1. In this review we will discuss the relationship between biological programs designed to regulate recombinase function.


Asunto(s)
Recombinación Homóloga , Meiosis , Proteínas de Ciclo Celular/metabolismo , Segregación Cromosómica , Proteínas de Unión al ADN/metabolismo , Recombinasa Rad51/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
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