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Mgm101 is a Rad52-related protein required for mitochondrial DNA recombination.

ABSTRACT: Homologous recombination is a conserved molecular process that has primarily evolved for the repair of double-stranded DNA breaks and stalled replication forks. However, the recombination machinery in mitochondria is poorly understood. Here, we show that the yeast mitochondrial nucleoid protein, Mgm101, is related to the Rad52-type recombination proteins that are widespread in organisms from bacteriophage to humans. Mgm101 is required for repeat-mediated recombination and suppression of mtDNA fragmentation in vivo. It preferentially binds to single-stranded DNA and catalyzes the annealing of ssDNA precomplexed with the mitochondrial ssDNA-binding protein, Rim1. Transmission electron microscopy showed that Mgm101 forms large oligomeric rings of ∼14-fold symmetry and highly compressed helical filaments. Specific mutations affecting ring formation reduce protein stability in vitro. The data suggest that the ring structure may provide a scaffold for stabilization of Mgm101 by preventing the aggregation of the otherwise unstable monomeric conformation. Upon binding to ssDNA, Mgm101 is remobilized from the rings to form distinct nucleoprotein filaments. These studies reveal a recombination protein of likely bacteriophage origin in mitochondria and support the notion that recombination is indispensable for mtDNA integrity.

SUBMITTER: Mbantenkhu M

PROVIDER: S-EPMC3234957 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Mgm101: A double-duty Rad52-like protein.

Project description:Mgm101 has well-characterized activity for the repair and replication of the mitochondrial genome. Recent work has demonstrated a further role for Mgm101 in nuclear DNA metabolism, contributing to an S-phase specific DNA interstrand cross-link repair pathway that acts redundantly with a pathway controlled by Pso2 exonuclease. Due to involvement of FANCM, FANCJ and FANCP homologues (Mph1, Chl1 and Slx4), this pathway has been described as a Fanconi anemia-like pathway. In this pathway, Mgm101 physically interacts with the DNA helicase Mph1 and the MutS? (Msh2/Msh6) heterodimer, but its precise role is yet to be elucidated. Data presented here suggests that Mgm101 functionally overlaps with Rad52, supporting previous suggestions that, based on protein structure and biochemical properties, Mgm101 and Rad52 belong to a family of proteins with similar function. In addition, our data shows that this overlap extends to the function of both proteins at telomeres, where Mgm101 is required for telomere elongation during chromosome replication in rad52 defective cells. We hypothesize that Mgm101 could, in Rad52-like manner, preferentially bind single-stranded DNAs (such as at stalled replication forks, broken chromosomes and natural chromosome ends), stabilize them and mediate single-strand annealing-like homologous recombination event to prevent them from converting into toxic structures.

| S-EPMC5176325 | biostudies-literature

A properly configured ring structure is critical for the function of the mitochondrial DNA recombination protein, Mgm101.

Project description:Mgm101 is a Rad52-type recombination protein of bacteriophage origin required for the repair and maintenance of mitochondrial DNA (mtDNA). It forms large oligomeric rings of ?14-fold symmetry that catalyze the annealing of single-stranded DNAs in vitro. In this study, we investigated the structural elements that contribute to this distinctive higher order structural organization and examined its functional implications. A pair of vicinal cysteines, Cys-216 and Cys-217, was found to be essential for mtDNA maintenance. Mutations to the polar serine, the negatively charged aspartic and glutamic acids, and the hydrophobic amino acid alanine all destabilize mtDNA in vivo. The alanine mutants have an increased propensity of forming macroscopic filaments. In contrast, mutations to aspartic acid drastically destabilize the protein and result in unstructured aggregates with severely reduced DNA binding activity. Interestingly, the serine mutants partially disassemble the Mgm101 rings into smaller oligomers. In the case of the C216S mutant, a moderate increase in DNA binding activity was observed. By using small angle x-ray scattering analysis, we found that Mgm101 forms rings of ?200 ? diameter in solution, consistent with the structure previously established by transmission electron microscopy. We also found that the C216A/C217A double mutant tends to form broken rings, which likely provide free ends for seeding the growth of the super-stable but functionally defective filaments. Taken together, our data underscore the importance of a delicately maintained ring structure critical for Mgm101 activity. We discuss a potential role of Cys-216 and Cys-217 in regulating Mgm101 function and the repair of damaged mtDNA under stress conditions.

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| S-EPMC8861003 | biostudies-literature

RAD52 is required for RNA-templated recombination repair in post-mitotic neurons.

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A short carboxyl-terminal tail is required for single-stranded DNA binding, higher-order structural organization, and stability of the mitochondrial single-stranded annealing protein Mgm101.

Project description:Mgm101 is a Rad52-type single-stranded annealing protein (SSAP) required for mitochondrial DNA (mtDNA) repair and maintenance. Structurally, Mgm101 forms large oligomeric rings. Here we determine the function(s) of a 32-amino acid carboxyl-terminal tail (Mgm101(238-269)) conserved in the Mgm101 family of proteins. Mutagenic analysis shows that Lys-253, Trp-257, Arg-259, and Tyr-268 are essential for mtDNA maintenance. Mutations in Lys-251, Arg-252, Lys-260, and Tyr-266 affect mtDNA stability at 37°C and under oxidative stress. The Y268A mutation severely affects single-stranded DNA (ssDNA) binding without altering the ring structure. Mutations in the Lys-251-Arg-252-Lys-253 positive triad also affect ssDNA binding. Moreover, the C-tail alone is sufficient to mediate ssDNA binding. Finally, we find that the W257A and R259A mutations dramatically affect the conformation and oligomeric state of Mgm101. These structural alterations correlate with protein degradation in vivo. The data thus indicate that the C-tail of Mgm101, likely displayed on the ring surface, is required for ssDNA binding, higher-order structural organization, and protein stability. We speculate that an initial electrostatic and base-stacking interaction with ssDNA could remodel ring organization. This may facilitate the formation of nucleoprotein filaments competent for mtDNA repair. These findings could have broad implications for understanding how SSAPs promote DNA repair and genome maintenance.

| S-EPMC3655812 | biostudies-literature

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| S-EPMC3093836 | biostudies-other

Mapping mitochondrial DNA recombination in yeast

Project description:In this study, we present the first genome-wide recombination map for mitochondrial DNA in yeast. We also assess the impact of the genetic background and of several gene deletions on the recombination profiles.

2014-10-22 | E-MTAB-2474 | biostudies-arrayexpress

The structure and DNA-binding properties of Mgm101 from a yeast with a linear mitochondrial genome.

Project description:To study the mechanisms involved in the maintenance of a linear mitochondrial genome we investigated the biochemical properties of the recombination protein Mgm101 from Candida parapsilosis. We show that CpMgm101 complements defects associated with the Saccharomyces cerevisiae mgm101-1(ts) mutation and that it is present in both the nucleus and mitochondrial nucleoids of C. parapsilosis. Unlike its S. cerevisiae counterpart, CpMgm101 is associated with the entire nucleoid population and is able to bind to a broad range of DNA substrates in a non-sequence specific manner. CpMgm101 is also able to catalyze strand annealing and D-loop formation. CpMgm101 forms a roughly C-shaped trimer in solution according to SAXS. Electron microscopy of a complex of CpMgm101 with a model mitochondrial telomere revealed homogeneous, ring-shaped structures at the telomeric single-stranded overhangs. The DNA-binding properties of CpMgm101, together with its DNA recombination properties, suggest that it can play a number of possible roles in the replication of the mitochondrial genome and the maintenance of its telomeres.

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Project description:Replication of herpes simplex virus 1 is coupled to recombination, but the molecular mechanisms underlying this process are poorly characterized. The role of Rad51 and Rad52 recombinases in viral recombination was examined in human fibroblast cells 1BR.3.N (wild type) and in GM16097 with replication defects caused by mutations in DNA ligase I. Intermolecular recombination between viruses, tsS and tsK, harboring genetic markers gave rise to ?17% recombinants in both cell lines. Knock-down of Rad51 and Rad52 by siRNA reduced production of recombinants to 11% and 5%, respectively, in wild type cells and to 3% and 5%, respectively, in GM16097 cells. The results indicate a specific role for Rad51 and Rad52 in recombination of replicating herpes simplex virus 1 DNA. Mixed infections using clinical isolates with restriction enzyme polymorphisms in the US4 and US7 genes revealed recombination frequencies of 0.7%/kbp in wild type cells and 4%/kbp in GM16097 cells. Finally, tandem repeats in the US7 gene remained stable upon serial passage, indicating a high fidelity of recombination in infected cells.

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Human RAD52 Captures and Holds DNA Strands, Increases DNA Flexibility, and Prevents Melting of Duplex DNA: Implications for DNA Recombination.

Project description:Human RAD52 promotes annealing of complementary single-stranded DNA (ssDNA). In-depth knowledge of RAD52-DNA interaction is required to understand how its activity is integrated in DNA repair processes. Here, we visualize individual fluorescent RAD52 complexes interacting with single DNA molecules. The interaction with ssDNA is rapid, static, and tight, where ssDNA appears to wrap around RAD52 complexes that promote intra-molecular bridging. With double-stranded DNA (dsDNA), interaction is slower, weaker, and often diffusive. Interestingly, force spectroscopy experiments show that RAD52 alters the mechanics dsDNA by enhancing DNA flexibility and increasing DNA contour length, suggesting intercalation. RAD52 binding changes the nature of the overstretching transition of dsDNA and prevents DNA melting, which is advantageous for strand clamping during or after annealing. DNA-bound RAD52 is efficient at capturing ssDNA in trans. Together, these effects may help key steps in DNA repair, such as second-end capture during homologous recombination or strand annealing during RAD51-independent recombination reactions.

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