Mechanism of homologous recombination and implications for aging-related deletions in mitochondrial DNA

Abstract

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.

Fig 1

Simplified schematic of the early steps in a canonical homologous recombination pathway in the eukaryotic nucleus and in bacteria. Upon DNA damage, the free ends of a DSB are first processed by an exonuclease. The exposed 3′ ssDNA tails are coated by single-strand DNA binding proteins (SSBs) to prevent the formation of secondary structures. The recombination mediator, Rad52 (or RecO in bacteria), then displaces SSB and recruits the Rad51/RecA-type recombinase to form presynaptic helical nucleoprotein filaments. These filaments then initiate homology search and catalyze ATP-dependent strand invasion within duplex DNA templates. The invading strand provides a free 3′ end for priming DNA replication that allows the restoration of genetic information missing from the dsDNA breaks. The Rad52 protein has a second function in this pathway, which is to capture the second end through its single-strand annealing activity. After DNA synthesis and ligation, double Holliday junctions are formed. The Holliday junctions can be resolved by different molecular strategies with or without DNA strand crossover. Without the capturing of the second end by Rad52, the invading strand may be dissociated from the D-loop and reannealed to the ssDNA on the second end, a process known as synthesis-dependent strand annealing (not shown).

Fig 2

Sequence conservation among closely related Mgm101 orthologs from the fungal (Saccharomyces cerevisiae, Kluyveromyces lactis, Schizosaccharomyces pombe, Aspergillus nidulans, Neurospora crassa, and Ustilago maydis), animal (Nematostella vectensis), and Dictyostelids (Dictylostelium discoideum) lineages within the unikont clade of the eukaryotic tree. Highlighted are the residues analyzed by mutagenesis in the S. cerevisiae Mgm101. The P162S and D129N alleles are temperature sensitive for mtDNA maintenance in vivo (157, 159). N150, F153, and F235 are conserved in Rad52, and their replacement by alanine affects protein folding and destabilize mtDNA (146). Similar mutations in Rad52 affect DNA repair in the nucleus. The C216/C217 cysteine pair is speculated to sense the redox state or other types of signals in mitochondria, which regulates Mgm101 activity (167). The K251/R252/K253 triad, Y266, and Y268 are required for ssDNA binding, and R259 is essential for the maintenance of the ring structure (179).

Fig 3

Molecular modeling of the Mgm101 core domain (from A115 to V237) based on the Rad52 structure (1H2I). (A) The positions of N150, F153, and F235, which are highly conserved in Rad52-related proteins, are highlighted. Also indicated are C216 and C217, which form a putative redox sensor for regulating Mgm101 function. (B) Surface representation model of a 14-mer ring formed by Mgm101^115-237. (Adapted from reference 165 [© The American Society for Biochemistry and Molecular Biology].)

Fig 4

A model for activation of Mgm101 for mtDNA repair. The Mgm101 rings are proposed to be the storage form of the protein in mt-nucleoids. In response to oxidative stress or other signals, the C216/C217 cysteine pair is modified. This may induce structural remodeling in the rings, which stimulates ssDNA binding and the repair of dsDNA breaks.

Fig 5

Confocal microscopy showing that overexpressed Mgm101-GFP forms bright structures resembling the Rad52 foci in the nuclear DNA repair center. Mgm101-GFP was expressed from the constitutive ADH1 promoter. Scale bar, 5 μm. DIC, differential interference contrast.

Fig 6

Structural organization of Mgm101 in comparison with the nuclear Rad52, the Sak protein from the lactococcal phage ul36, and the mitochondrial Rad52-1B variant of A. thaliana. (A) Rad52 is a central recombination protein for mediating Rad51-catalyzed strand invasion of dsDNA in the nucleus. It has retained the single-strand annealing (SSA) activity on its N terminus. Rad52 has acquired a large C-terminal domain that interacts with the nuclear single-strand binding protein RPA. It recruits Rad51 onto ssDNA and enhances Rad51 nucleation through a second DNA binding activity (–233). The yeast Rad52 paralog, Rad59, lacks the C-terminal Rad51-interacting domain (234). (B) The higher-order structural organization of Mgm101 in comparison with Sak and the SSA domain of Rad52. (a to c) Transmission electron microscopy shows that Mgm101 forms rings of ∼14-fold symmetry (a) and that Sak (b) and Rad52 (c) form rings with 11 subunits. These rings have diameters of 200 Å for Mgm101, 150 Å for Sak, and 130 Å for the SSA domain of Rad52. (d and e) Mgm101 and Sak, but not Rad52, also form highly compressed filaments with pitches of only 50 and 55 Å, respectively. (f to h) In the presence of ssDNA, Mgm101 and Sak form condensed nucleoprotein complexes, in contrast to Rad52, which forms uniform thin filaments on ssDNA. (Panels a, d, and f are reprinted from reference with permission of the publisher [© The American Society for Biochemistry and Molecular Biology]. Panels b, c, e, and g are reprinted from reference with permission of Elsevier. Panel h is adapted from reference with permission of the publisher [© The American Society for Biochemistry and Molecular Biology].)

Fig 7

Models for the operation mode of a recombination system of bacteriophage origin in mitochondria and its implications for the generation of mtDNA deletions in aged cells. A single-stranded DNA-annealing protein such as Mgm101 plays a central role in the repair of double-strand DNA breaks (middle panel). After the resection of a dsDNA end by a 5′-3′ exonuclease, SSB is bound to the exposed ssDNA to prevent the formation of secondary structures. SSB is then displaced by an SSAP like Mgm101. The SSAP/ssDNA nucleoprotein filaments initiate recombination by annealing to homologous single-stranded DNA on the lagging strand of a replication fork, like the Redβ protein in the phage λ (172, 173). The Mgt1/Cce1 endonuclease may process the recombination junction before the loading of the mtDNA replisome. However, recombinational errors may occasionally occur, either by a classic single-strand annealing between intramolecularly repeated sequences, which generates unrepairable mtDNA deletions (right panel), or by homeologous annealing to a nonhomologous lagging strand, which causes mtDNA deletions and rearrangements (left panel). The mismatch repair protein Msh1 may play a role in recombination editing by rejecting homeologous annealing. In young cells, mtDNA recombination is kept at a very low level to clear the rarely arising DSBs. In aged cells, mtDNA recombination increases as a result of oxidative stress and elevated mtDNA damage. This may inevitably increase erroneous recombinational events, which leads to the time-dependent accumulation of unfixable deleted and rearranged mtDNAs.

Fig 8

A vicious-cycle model for homeologous recombination-mediated mtDNA deletions in aged cells. See the text for details.