A Short Carboxyl-terminal Tail is Required for ssDNA-binding, Higher Order Structural Organization and Stability of the Mitochondrial Single Strand Annealing Protein Mgm101.

Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, Syracuse, NY 13210, USA.
Molecular biology of the cell (Impact Factor: 4.47). 03/2013; 24(10). DOI: 10.1091/mbc.E13-01-0006
Source: PubMed


Mgm101 is a Rad52-type Single Strand Annealing Protein (SSAP) required for mitochondrial DNA (mtDNA) repair and maintenance. Structurally, Mgm101 forms large oligomeric rings. Here, we determined the function(s) of a 32-amino acid carboxyl-terminal tail (Mgm101(238-269)) conserved in the Mgm101-family of proteins. Mutagenic analysis showed that Lys253, Trp257, Arg259 and Tyr268 are essential for mtDNA maintenance. Mutations in Lys251, Arg252, Lys260 and Tyr266 affect mtDNA stability at 37°C and under oxidative stress. The Y268A mutation severely affects ssDNA-binding without altering the ring structure. Mutations in the Lys251-Arg252-Lys253 positive triad also affect ssDNA-binding. Moreover, we found that the C-tail alone is sufficient to mediate ssDNA-binding. Finally, we found that the W257A and R259A mutations dramatically affect the conformation and oligomeric state of Mgm101. These structural alterations correlated 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.

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Available from: Sara Wierzbicki, Mar 26, 2015
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    ABSTRACT: SUMMARY Homologous recombination is a universal process, conserved from bacteriophage to human, which is important for the repair of double-strand DNA breaks. Recombination in mitochondrial DNA (mtDNA) was documented more than 4 decades ago, but the underlying molecular mechanism has remained elusive. Recent studies have revealed the presence of a Rad52-type recombination system of bacteriophage origin in mitochondria, which operates by a single-strand annealing mechanism independent of the canonical RecA/Rad51-type recombinases. Increasing evidence supports the notion that, like in bacteriophages, mtDNA inheritance is a coordinated interplay between recombination, repair, and replication. These findings could have profound implications for understanding the mechanism of mtDNA inheritance and the generation of mtDNA deletions in aging cells.
    No preview · Article · Sep 2013 · Microbiology and molecular biology reviews: MMBR