A mutant form of Dmc1 that bypasses the requirement for accessory protein Mei5-Sae3 reveals independent activities of Mei5-Sae3 and Rad51 in Dmc1 filament stability

Autor: Douglas K. Bishop, Diedre Reitz, Jennifer Grubb
Jazyk: angličtina
Rok vydání: 2019
Předmět:
Cancer Research
Chromosomal Proteins
Non-Histone
RAD51
Cell Cycle Proteins
Artificial Gene Amplification and Extension
Yeast and Fungal Models
QH426-470
Biochemistry
Chromosomal crossover
0302 clinical medicine
Mutant protein
Recombinase
DNA Breaks
Double-Stranded
Crossing Over
Genetic
Cell Cycle and Cell Division
DNA
Fungal
Homologous Recombination
Genetics (clinical)
0303 health sciences
Chemistry
Protein Stability
Chromosome Biology
Eukaryota
Recombinant Proteins
Cell biology
Nucleic acids
Meiosis
Experimental Organism Systems
Cell Processes
Gain of Function Mutation
Research Article
Saccharomyces cerevisiae Proteins
DNA repair
DNA recombination
Saccharomyces cerevisiae
Biology
Research and Analysis Methods
Models
Biological
Recombinases
03 medical and health sciences
Saccharomyces
Model Organisms
DNA-binding proteins
Homologous chromosome
Genetics
Sister chromatids
Ectopic recombination
Molecular Biology Techniques
Molecular Biology
Ecology
Evolution
Behavior and Systematics
030304 developmental biology
fungi
Organisms
Fungi
Biology and Life Sciences
Proteins
DNA
Cell Biology
Yeast
Amino Acid Substitution
Animal Studies
DMC1
Rad51 Recombinase
Homologous recombination
Recombinase Polymerase Amplification
030217 neurology & neurosurgery
Zdroj: PLoS Genetics, Vol 15, Iss 12, p e1008217 (2019)
PLoS Genetics
ISSN: 1553-7404
1553-7390
Popis: During meiosis, homologous recombination repairs programmed DNA double-stranded breaks. Meiotic recombination physically links the homologous chromosomes (“homologs”), creating the tension between them that is required for their segregation. The central recombinase in this process is Dmc1. Dmc1’s activity is regulated by its accessory factors including the heterodimeric protein Mei5-Sae3 and Rad51. We use a gain-of-function dmc1 mutant, dmc1-E157D, that bypasses Mei5-Sae3 to gain insight into the role of this accessory factor and its relationship to mitotic recombinase Rad51, which also functions as a Dmc1 accessory protein during meiosis. We find that Mei5-Sae3 has a role in filament formation and stability, but not in the bias of recombination partner choice that favors homolog over sister chromatids. Analysis of meiotic recombination intermediates suggests that Mei5-Sae3 and Rad51 function independently in promoting filament stability. In spite of its ability to load onto single-stranded DNA and carry out recombination in the absence of Mei5-Sae3, recombination promoted by the Dmc1 mutant is abnormal in that it forms foci in the absence of DNA breaks, displays unusually high levels of multi-chromatid and intersister joint molecule intermediates, as well as high levels of ectopic recombination products. We use super-resolution microscopy to show that the mutant protein forms longer foci than those formed by wild-type Dmc1. Our data support a model in which longer filaments are more prone to engage in aberrant recombination events, suggesting that filament lengths are normally limited by a regulatory mechanism that functions to prevent recombination-mediated genome rearrangements.
Author summary During meiosis, two rounds of division follow a single round of DNA replication to create the gametes for biparental reproduction. The first round of division requires that the homologous chromosomes become physically linked to one another to create the tension that is necessary for their segregation. This linkage is achieved through DNA recombination between the two homologous chromosomes, followed by resolution of the recombination intermediate into a crossover. Central to this process is the meiosis-specific recombinase Dmc1, and its accessory factors, which provide important regulatory functions to ensure that recombination is accurate, efficient, and occurs predominantly between homologous chromosomes, and not sister chromatids. To gain insight into the regulation of Dmc1 by its accessory factors, we mutated Dmc1 such that it was no longer dependent on its accessory factor Mei5-Sae3. Our analysis reveals that Dmc1 accessory factors Mei5-Sae3 and Rad51 have independent roles in stabilizing Dmc1 filaments. Furthermore, we find that although Rad51 is required for promoting recombination between homologous chromosomes, Mei5-Sae3 is not. Lastly, we show that our Dmc1 mutant forms abnormally long filaments, and high levels of aberrant recombination intermediates and products. These findings suggest that filaments are actively maintained at short lengths to prevent deleterious genome rearrangements.
Databáze: OpenAIRE
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