The mechanism by which SSAPs catalyze SSA is not well understood. Among the pertinent questions are the following: 1) How is ssDNA binding initiated? 2) Is the ring structure required for ssDNA binding? 3) How is the homology search promoted? Studies of the eukaryotic Rad52 protein support the model that ssDNA binds to the central groove along the surface of the ring (Singleton et al., 2002
). Homology search is believed to proceed by successive interaction between two separate ssDNA-wrapped rings until the formation of a stable duplex. The force generated by the zippering of annealed DNA duplexes may drive ssDNA release from the overlapping nucleoprotein complexes (Rothenberg et al., 2008
; Grimme et al., 2010
). However, it remains perplexing that Rad52 often forms nucleoprotein filaments of 10-nm thickness on ssDNA, which suggests that the ring structure is disrupted upon DNA binding (Kagawa et al., 2001
). Although several mutations in the presumptive central binding groove affect ssDNA-binding activity (Kagawa et al., 2002
; Lloyd et al., 2005
), these data need to be validated by solving the structure of Rad52/ssDNA cocrystals. On the other hand, study of Redβ by atomic force microscopy revealed that the ring/helix organization is clearly disrupted when the phage protein interacts with ssDNA (Erler et al., 2009
). In this case, monomeric structures are also detected in the Redβ–ssDNA complexes. It was proposed that it is the interaction between two ssDNA-bound Redβ monomers that facilitates the annealing of the ssDNA molecules. The latter observations point to the possibility that the SSAPs from various sources may have different modes of action in binding to ssDNA and in catalyzing strand annealing.
Mgm101 forms condensed nucleoprotein filaments on ssDNA, like the bacteriophage SSAPs, such as the Sak protein from the lactococcal phage ul36 (Ploquin et al., 2008
; Mbantenkhu et al., 2011
). It also forms highly compressed helical filaments, like Redβ from the bacteriophage λ (Passy et al., 1999
). These molecular features are not seen in Rad52. The data from the present study further suggest that Mgm101, and probably also the bacteriophage SSAPs, may have a DNA-binding mode different from that proposed for Rad52. Mutation in Tyr-65 of Rad52 reduces ssDNA binding by fourfold (Kagawa et al., 2002
; Lloyd et al., 2005
). Tyr-65 is the only conserved residue in several SSAPs among the amino acids predicted to bind to ssDNA in the putative central binding groove. This residue corresponds to Tyr-139 in Mgm101 and Tyr-42 in Sak ( and ). We provided evidence that Tyr-139 is essential for the mtDNA maintenance function of Mgm101 in vivo, but the Y139A mutation does not affect ssDNA binding in vitro. The similar Y42A mutation in Sak was also been to have little effect on DNA binding under similar conditions (Ploquin et al., 2008
). It is noteworthy that Tyr-139 is not conserved in some SSAPs, including Redβ (Lopes et al., 2010
). These observations raise the possibility that the central core of Mgm101 may not play a major role, if any, in ssDNA binding. Mgm101 may have evolved a novel mode of interaction with ssDNA substrates.
Mutagenesis analysis combined with in vivo functional assay and in vitro biochemical characterization revealed a robust ssDNA-binding site at the short C-tail. The C-tail is highly conserved among the Mgm101 homologues that function in mitochondria (). This particular domain of 32 amino acids in length is not conserved in Rad52, which operates in the eukaryotic nucleus. The C-tail contains highly conserved aromatic and basic amino acids that are predicted to form two β-sheets followed by an unstructured end. Among the 10 amino acids analyzed, Lys-253, Trp-257, Arg-259, and Tyr-268 were found to be essential for mtDNA maintenance in vivo. Mutations in Lys-251, Lys-252, Lys-260, and Tyr-266 affected mtDNA maintenance only under stress conditions. Petite colony formation was dramatically increased in these mutants when cells were incubated at high temperature (37°C). Increased petite formation was also seen when cells were treated with hydrogen peroxide, which is consistent with the previously reported role of Mgm101 in repair of oxidatively damaged mtDNA (Meeusen et al., 1999
). The most intriguing finding is perhaps that mutations in some of these residues affect ssDNA binding. We found that the gross ring structure of Mgm101Y268A
, and Mgm101(K251-K253)A
is unaltered, as confirmed by single-particle imaging using transmission electron microscopy. There is apparently an increase in lateral interaction between the rings, which changes the elution profile of the proteins in size exclusion chromatography. However, ssDNA binding by these mutant proteins is severely affected. ssDNA-binding activity of Mgm101Y268A
, which eluted mainly like the wild type in size exclusion chromatography, is reduced ninefold. ssDNA-binding activity of Mgm101K253A
is reduced 5.5-fold. ssDNA-binding activity of the triple substitution mutant Mgm101(K251-K253)A
is reduced 8.2-fold, indicating that Lys-251 and/or Arg-252 also contribute to the interaction with ssDNA. A confirmation for the ssDNA-binding activity of the C-tail comes from an experiment showing that the C-tail alone, when fused to MBP, is sufficient to mediate ssDNA binding. The data indicate that the C-tail of Mgm101 is capable of binding to ssDNA, which could be critical for mtDNA repair and maintenance in vivo. Although the in vitro data suggest that the 251–KRK–253 triad may contribute to the interaction with ssDNA, this may not fully account for the defect in mtDNA maintenance in vivo (). The Mgm101K253A
proteins are partially unstable in vivo. It is possible that ssDNA-binding defect and protein instability both contribute to mtDNA instability. We previously reported the C240A mutation in the C-tail. This mutation does not affect mtDNA maintenance in vivo (Mbantenkhu et al., 2011
), and the ssDNA-binding activity of the mutant protein is not affected compared with the wild type (our unpublished data).
The mechanism of ssDNA binding has been extensively studied in oligonucleotide/oligosaccharide-binding fold (OB-fold) proteins. In these cases, ssDNA interacts with a rather limited surface on the proteins primarily via stacking interactions with aromatic amino acid chains (Theobald et al., 2003
). The base-stacking interactions allow the protein to distinguish ssDNA from dsDNA, whose base has less accessibility. Although Mgm101 does not belong to the OB-fold protein family, its C-tail may represent a novel module for ssDNA recognition. We speculate that Tyr-268 may play a pivotal role in mediating interaction with ssDNA by stacking interactions. The second aromatic residue, Tyr-266, which also affects mtDNA stability in response to high temperature and oxidative stress, may also promote stacking and assist the stabilization of the interactions with ssDNA. On the other hand, the 251–KRK–253 triad may attract ssDNA to the ring surface through electrostatic interactions, which facilitates its interactions with Tyr-268 and Tyr-266.
Biochemical analysis of Mgm101 with alanine substitution also revealed an important role of the Mgm101 C-tail in structural stabilization. Even in the MBP-fused form, mutation in Arg-259 resulted in an unstable protein that tends to elute in the void volume in size exclusion chromatography, which is suggestive of aggregate formation. The mutant protein after cleavage from MBP was not recoverable. A similar property was observed when Mgm101 lacking the entire C-tail was analyzed. In the latter case, a sizable MBP-fused monomeric peak was noticeable in addition to a major peak in the void volume. These data suggest that the C-tail is important for oligomerization and stabilization of the protein. Arg-259 may play a key role in mediating interactions between the adjacent subunits in the ring by facilitating salt bridge formation. The mutation may disassemble the rings into monomeric Mgm101, which is unstable in solution. A rapid conversion of monomeric Mgm101 to insoluble aggregates has been speculated (Mbantenkhu et al., 2011
; Nardozzi et al., 2012
). Although it can only be transiently detected, the monomeric peak of MBP-Mgm101ΔC is the only unoligomerized Mgm101 species detected so far. The presence of MBP likely contributes to the partial stabilization of the mutant form of monomeric Mgm101 in solution.
A role of the C-tail in maintaining an intact ring could be analogous to the C-terminal end of the SSA domain of Rad52 (or Rad521-209
). The C-terminal end of this truncated form of Rad52 is characterized by a flexible linker, which is followed the α-helix 5, which swaps across the subunit interface and interacts with an adjacent subunit in the undecameric ring (Kagawa et al., 2002
; Singleton et al., 2002
). This “helix-swapping” strategy promotes subunit interactions, thereby stabilizing the ring structure. Whether a similar strategy is used for oligomerization in the full-length heptameric Rad52 is unknown. In place of α-helix 5 in Rad521-209
, the C-tail of Mgm101 is predicted to form two β strands followed by a short, unstructured C-terminal end (β6 and β7; see ). These β strands are likely involved in interactions with adjacent subunits in the ring. Arg-259 may be required for stabilizing the β6–β7 interaction or for direct interaction with adjacent subunits. As a consequence, mutation in Arg-259 prevents ring formation.
Trp-257 is another aromatic amino acid on the C-tail of Mgm101 essential for its function. However, mutation in Trp-257 does not have a detectable defect in ssDNA binding. Mgm101W257A has a peculiar elution profile on the Superose 6 column. The protein was eluted as a sharp peak corresponding to the molecular weight of monomeric Mgm101. However, blue-native gel showed that the mutant protein forms oligomeric structures indistinguishable from the wild type. Inspection by transmission electron microscopy did not show an obvious difference in the shape and size of the rings formed by Mgm101W257A and the wild-type protein. Furthermore, Mgm101W257A also remained stable in solution like the wild type. It is likely that Mgm101W257A exists as oligomeric rings rather than in a monomeric form. It is possible that the W257A mutation may change the surface property of the rings, which increases interactions with the column matrix and causes the dramatic retardation of the protein on the column. This novel property is probably corrected or masked by the tagging of the 42-kDa MBP on the N-terminus, which permitted an elution profile close to that of the wild-type protein (see and ).
We observed that Mgm101R259A and Mgm101W257A not only are structurally unstable in vitro, but are also highly unstable in vivo. This provides an explanation for the rapid loss of mtDNA in the mutants. It seems that the maintenance of an appropriately configured ring structure is important for protein stability in vivo. Monomerization or changes to the ring surface properties may trigger protein degradation. For future studies, it would be interesting to determine which protease is involved in degrading disassembled and improperly assembled Mgm101 structures in the mtDNA nucleoids.
In summary, we found that the 32-aa C-tail of Mgm101 is required for ssDNA binding, structural organization, and protein stability in vivo. It can be speculated that the positively charged 251–KRK-253 triad may initiate the contact with the phosphate backbone of ssDNA by electrostatic interactions. The highly conserved 266–YPY–268 motif at the extreme end of the protein may stabilize the interactions by base stacking. These interactions could disrupt the ring structure maintained by salt bridge interactions involving Arg-259. This may facilitate the deployment of additional subunits in the ring to spread the interactions with ssDNA. The model is consistent with the idea that the ring structure serves as a store in the mitochondrial nucleoids so that the protein can be rapidly mobilized after mtDNA damage and the initial interaction with DNA. This ultimately generates recombination-competent nucleoprotein filaments for the repair of double-stranded breaks in mtDNA, in concert with other repair proteins, including those involved in homologous pairing and nonhomologous end joining (Ling et al., 1995
; Ling and Shibata, 2002
; Kalifa et al., 2012
). Future studies are necessary to test this model.