hMSH4-hMSH5 adenosine nucleotide processing and interactions with homologous recombination machinery

Abstract

We have previously demonstrated that the human heterodimeric meiosis-specific MutS homologs, hMSH4-hMSH5, bind uniquely to a Holliday Junction and its developmental progenitor (Snowden, T., Acharya, S., Butz, C., Berardini, M., and Fishel, R. (2004) Mol. Cell 15, 437-451). ATP binding by hMSH4-hMSH5 resulted in the formation of a sliding clamp that dissociated from the Holliday Junction crossover region embracing two duplex DNA arms. The loading of multiple hMSH4-hMSH5 sliding clamps was anticipated to stabilize the interaction between parental chromosomes during meiosis double-stranded break repair. Here we have identified the interaction region between the individual subunits of hMSH4-hMSH5 that are likely involved in clamp formation and show that each subunit of the heterodimer binds ATP. We have determined that ADP-->ATP exchange is uniquely provoked by Holliday Junction recognition. Moreover, the hydrolysis of ATP by hMSH4-hMSH5 appears to occur after the complex transits the open ends of model Holliday Junction oligonucleotides. Finally, we have identified several components of the double-stranded break repair machinery that strongly interact with hMSH4-hMSH5. These results further underline the function(s) and interactors of hMSH4-hMSH5 that ensure accurate chromosomal repair and segregation during meiosis.

FIGURE 1. The interaction regions between hMSH4 and hMSH5

A, regions of hMSH4 required for interaction with hMSH5. B, regions of hMSH5 required for interaction with hMSH4. [³⁵S]Met-labeled full-length and truncation mutants of IVTT-hMSH4 (A) or truncation mutants of IVTT-hMSH5 (B) were incubated with glutathione beads prebound with either GST (alone) or full-length GST-hMSH5 (A) or full-length GST-hMSH4. Following processing, reactions were separated on a 10% SDS-PAGE gel and visualized by phosphorimager. Illustrations of the mutant constructs are shown below the gels on both panels. Numbers correspond to the individual numbered deletions (del) and the reaction separated by the gel above. The consensus interaction peptides are shown in the shaded blue-gray (Int_rel 20–75%) or black (Int_rel 75–100%) box. C, hMSH4-hMSH5 forms a specific heterodimer that contains two interaction regions. An illustration with the two interaction regions containing numbered amino acids is shown.

FIGURE 2. Both subunits of hMSH4-hMSH5 bind ATP and ATPγS

hMSH4-hMSH5 (100 nm) was incubated with the indicated concentrations of labeled ATP and exposed to UV cross-linking. Reactions were separated on a 10% SDS-PAGE gel and visualized via phosphorimager (shown below each panel). A, [α-³²P]ATP; B, [γ-³²P]ATP; C, [γ-³⁵S]ATP;D, representative Coomassie-stained gel showing the position and equal loading of the hMSH4 and hMSH5; E, UV cross-linking of hMSH4-hMSH5 (100 nm) with [γ-³²P]ATP (1 µm) in the presence or absence of Holiday Junction DNA (200 nm). Relative phosphorimager quantitation is shown: ○, hMSH4; ●, hMSH5.

FIGURE 3. Competition of adenosine nucleotide cross-linking

hMSH4-hMSH5 (100 nm) was incubated with the labeled ATP (500 nm; see below), the indicated concentrations of unlabeled ATP (µm; shown in the boxes above lanes), and subjected to UV cross-linking. Reactions were separated on a 10% PAGE gel, visualized, and quantitated by phosphorimager (shown below each panel). A, [α-³²P]ATP (S_0.5(hMSH4) ≈ 4 µm; S_0.5(hMSH5) ≈ 4 µm); B, [γ-³²P]ATP (S_0.5(hMSH4) ≈ 3 µm; S_0.5(hMSH5) ≈ 12 µm); C, [γ-³⁵S]ATP (S_0.5(hMSH4) ≈ 7 µm; S_0.5(hMSH5) ≈ 7 µm). Relative phosphorimager quantitation is shown: ○, hMSH4; ●, hMSH5.

FIGURE 4. Holliday Junctions uniquely activate the hMSH4-hMSH5 steady-state ATPase and ADP → ATP exchange activities

A,DNA structure stimulation of the hMSH4-hMSH5 ATPase. Michaelis-Menten analysis of the hMSH4-hMSH5 (200 nm) ATPase activity was performed as previously described (11, 32) in the absence of DNA (▲), in the presence of duplex DNA (dsDNA, 600 nm; ●), Y-Junction DNA (YoJo, 600 nm; ♦), frayed Y-Junction DNA (Frayed-YoJo, 600 nm;), or Holliday Junction DNA (Hojo, 600 nm; ■) in a 20-µl reaction. Error bars indicate the standard deviation of at least three independent experiments. B,DNA structure provoked hMSH4-hMSH5 ADP → ATP exchange. hMSH4-hMSH5 was pre-bound to [³H]ADP followed by the addition of 500 µm unlabeled ATP in the absence of DNA (■), in the presence of 3-fold excess duplex DNA (dsDNA, ▲), Y-Junction DNA(YoJo, ♦), frayed Y-JunctionDNA(Frayed-YoJo,), and Holliday JunctionDNA(HoJo, ●). Results are plotted as the fraction of total [³H]ADP that remained bound over time. The standard of deviation was calculated from the average of at least four different concentrations of hMSH4-hMSH5 (25, 50, 75, and 100 nm) and from at least three independent experiments. C, diagram of substrates used in A and B.

FIGURE 5. Blocking the Holliday Junction ends does not affect ADP → ATP exchange but does inhibit the steady-state ATPase of hMSH4-hMSH5

A, ADP → ATP exchange. Pre-steady state ADP → ATP exchange was performed as described under “Experimental Procedures” with hMSH4-hMSH5 (200 nm) in the absence of DNA but including streptavidin (800 µg/ml, ■), in the presence of HoJo DNA containing a 3′-biotin on the four DNA ends (b-Hojo, 600 nm, ▲), or in the presence of b-Hojo DNA (600 nm) including streptavidin (800 µg/ml, streptavidin +b-HoJo; ●). Error bars indicate the standard deviation of at least three independent experiments, which in some cases is within the symbol size. B, hMSH4-hMSH5 ATPase activity. Michaelis-Menten analysis of the hMSH4-hMSH5 (200 nm) ATPase activity was performed as previously described (11, 32) in the presence of unbiotinylated HoJo DNA (600 nm) and including streptavidin (800 µg/ml; streptavidin + HoJo, ●), in the presence of HoJo DNA containing a 3′-biotin on the four DNA ends (600 nm; b-Hojo, ▲) or in the presence of b-Hojo DNA (600 nm) and including streptavidin (800 µg/ml; streptavidin+b-HoJo, ■). Error bars indicate the standard deviation of at least three independent experiments.

FIGURE 6. Blocking one duplex arm on both ends of a Holliday Junction stacked-duplex does not affect hMSH4-hMSH5 ADP → ATP exchange but inhibits ATPase activity

A, ADP → ATP exchange by hMSH4-hMSH5 is unaffected by the disposition of HoJo ends. Pre-steady state ADP → ATP exchange was performed as described under “Experimental Procedures” with hMSH4-hMSH5 (200 nm) in the absence of DNA (■) or in the presence of HoJo DNA (600 nm,●), single end-blocked stacked-duplex HoJo DNA (1EB, 600 nm, ♦), or double end-blocked stacked-duplex HoJo DNA (2EB, 600 nm, ▲). B, hMSH4-hMSH5 ATPase activity requires an open-end HoJo stacked duplex. Michaelis-Menten analysis of the hMSH4-hMSH5 (200 nm) ATPase activity was performed as previously described (11, 32) in the absence of DNA (▲) or in the presence of HoJo DNA (600 nm, ■), single end-blocked stacked-duplex HoJo DNA (1EB, 600 nm, ●), or double end-blocked stacked-duplex HoJo DNA (2EB, 600 nm,♦). Error bars indicate the standard deviation of at least three independent experiments.

FIGURE 7. Interactions between hMSH4 and hMSH5 with homologous recombination machinery

A, interaction of DSB repair proteins with hMSH5. [³⁵S]Methionine-labeled IVTT proteins were quantitated relative to the number of methionine residues in each protein and equimolar amounts were precipitated with an excess of purified GST-hMSH4 protein. GST alone was used as a precipitation control. Upper panel shows the gel of precipitated IVTT proteins. Precipitated material was quantitated by phosphorimager, nonspecific GST-only interaction subtracted, and the remaining interaction calculated relative to the known interactor hMSH5 (Int_rel(hMSH5)) and plotted in the lower panel. B, interaction of DSB repair proteins with hMSH4. [³⁵S]Methionine-labeled IVTT proteins were quantitated relative to the number of methionine residues in each protein and equimolar amounts precipitated with an excess of purified GST-hMSH5 protein. GST alone was used as a precipitation control. Upper panel shows the gel of precipitated IVTT proteins. Precipitated material was quantitated by phosphorimager, nonspecific GST-only interaction subtracted, the remaining interaction calculated relative to the known interactor hMSH4 (Int_rel(hMSH4)) and plotted in the lower panel