Limits...
Crystal structure of Hop2-Mnd1 and mechanistic insights into its role in meiotic recombination.

Kang HA, Shin HC, Kalantzi AS, Toseland CP, Kim HM, Gruber S, Peraro MD, Oh BH - Nucleic Acids Res. (2015)

Bottom Line: One end of the rod is linked to two juxtaposed winged-helix domains, and the other end is capped by extra α-helices to form a helical bundle-like structure.Deletion analysis shows that the helical bundle-like structure is sufficient for interacting with the Dmc1-ssDNA nucleofilament, and molecular modeling suggests that the curved rod could be accommodated into the helical groove of the nucleofilament.Remarkably, the winged-helix domains are juxtaposed at fixed relative orientation, and their binding to DNA is likely to perturb the base pairing according to molecular simulations.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, KAIST Institute for the Biocentury, Cancer Metastasis Control Center, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea.

Show MeSH

Related in: MedlinePlus

The C-terminal portion of Hop2–Mnd1 interacts with Dmc1 nucleofilament. (A) Fitting of the coiled coil of Hop2–Mnd1 into the helical groove of the Dmc1-ssDNA filament (EBI entry: EMD-1492). Surface of both Hop2–Mnd1 (blue) and human Dmc1-ssDNA filament (gray) are shown in mesh representation. (B) LZ3wCH of Hop2–Mnd1 is necessary for binding to Dmc1 nucleofilament. (Left) Schematic representation of the exonuclease I protection assay. (Right, top) Wild-type Hop2–Mnd1 and the indicated deletion mutants (2.5 μM) were individually incubated with Dmc1 nucleofilament and their ssDNA protection ability was analyzed by electrophoresis on a 15% native gel. (Right, bottom) The intensities of unreacted ssDNA relative to input ssDNA are shown. The experiment was performed in triplicate. (C) Mapping of conserved residues on the surface of Hop2–Mnd1.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4402518&req=5

Figure 6: The C-terminal portion of Hop2–Mnd1 interacts with Dmc1 nucleofilament. (A) Fitting of the coiled coil of Hop2–Mnd1 into the helical groove of the Dmc1-ssDNA filament (EBI entry: EMD-1492). Surface of both Hop2–Mnd1 (blue) and human Dmc1-ssDNA filament (gray) are shown in mesh representation. (B) LZ3wCH of Hop2–Mnd1 is necessary for binding to Dmc1 nucleofilament. (Left) Schematic representation of the exonuclease I protection assay. (Right, top) Wild-type Hop2–Mnd1 and the indicated deletion mutants (2.5 μM) were individually incubated with Dmc1 nucleofilament and their ssDNA protection ability was analyzed by electrophoresis on a 15% native gel. (Right, bottom) The intensities of unreacted ssDNA relative to input ssDNA are shown. The experiment was performed in triplicate. (C) Mapping of conserved residues on the surface of Hop2–Mnd1.

Mentions: In the Hop2–Mnd1 structure, the orientations of the three leucine zippers are different. Intriguingly, we found that the three leucine zippers in their respective orientations can be fitted into the helical groove in the filament of the Dmc1-ssDNA complex (Figure 6A) (44). While LZ1 and LZ2 could be snugly fitted into the groove, LZ3wCH having the capping helices appeared to cause some steric crash. In order to elaborate this observation, we produced five different deletion mutants and performed Exo I protection assays (Figure 6B). Cleavage of Dmc1-bound ssDNA by Exo I nuclease was suppressed by wild-type Hop2–Mnd1. Importantly, deletion constructs, which retain the LZ3wCH region (Figure 6B; ΔWHD, LZ2+LZ3wCH, LZ3wCH), exhibited a similar level of protection as wild type. In contrast, mutants lacking this region (Figure 6B; WHD+LZ1, ΔLZ3wCH) were far less protective. Thus, contrary to our expectation, the LZ3wCH region alone was sufficient for interacting with the Dmc1 nucleofilament. Induced-fit binding may be necessary for the interaction between LZ3wCH and the groove of the Dmc1 nucleofilament. Consistent with this notion, a proteolysis assay showed that mouse Hop2–Mnd1 complex affects the conformation of human Rad51 (45). Upon binding of LZ3wCH to the Dmc1 nucleofilament, LZ1 and LZ2 may passively position into the helical groove, and the flexibility of the LZ1–LZ2 and LZ2–LZ3 junctions may allow for their adjustments to the induced-fit conformational change of the Dmc1 nucleofilament.


Crystal structure of Hop2-Mnd1 and mechanistic insights into its role in meiotic recombination.

Kang HA, Shin HC, Kalantzi AS, Toseland CP, Kim HM, Gruber S, Peraro MD, Oh BH - Nucleic Acids Res. (2015)

The C-terminal portion of Hop2–Mnd1 interacts with Dmc1 nucleofilament. (A) Fitting of the coiled coil of Hop2–Mnd1 into the helical groove of the Dmc1-ssDNA filament (EBI entry: EMD-1492). Surface of both Hop2–Mnd1 (blue) and human Dmc1-ssDNA filament (gray) are shown in mesh representation. (B) LZ3wCH of Hop2–Mnd1 is necessary for binding to Dmc1 nucleofilament. (Left) Schematic representation of the exonuclease I protection assay. (Right, top) Wild-type Hop2–Mnd1 and the indicated deletion mutants (2.5 μM) were individually incubated with Dmc1 nucleofilament and their ssDNA protection ability was analyzed by electrophoresis on a 15% native gel. (Right, bottom) The intensities of unreacted ssDNA relative to input ssDNA are shown. The experiment was performed in triplicate. (C) Mapping of conserved residues on the surface of Hop2–Mnd1.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4402518&req=5

Figure 6: The C-terminal portion of Hop2–Mnd1 interacts with Dmc1 nucleofilament. (A) Fitting of the coiled coil of Hop2–Mnd1 into the helical groove of the Dmc1-ssDNA filament (EBI entry: EMD-1492). Surface of both Hop2–Mnd1 (blue) and human Dmc1-ssDNA filament (gray) are shown in mesh representation. (B) LZ3wCH of Hop2–Mnd1 is necessary for binding to Dmc1 nucleofilament. (Left) Schematic representation of the exonuclease I protection assay. (Right, top) Wild-type Hop2–Mnd1 and the indicated deletion mutants (2.5 μM) were individually incubated with Dmc1 nucleofilament and their ssDNA protection ability was analyzed by electrophoresis on a 15% native gel. (Right, bottom) The intensities of unreacted ssDNA relative to input ssDNA are shown. The experiment was performed in triplicate. (C) Mapping of conserved residues on the surface of Hop2–Mnd1.
Mentions: In the Hop2–Mnd1 structure, the orientations of the three leucine zippers are different. Intriguingly, we found that the three leucine zippers in their respective orientations can be fitted into the helical groove in the filament of the Dmc1-ssDNA complex (Figure 6A) (44). While LZ1 and LZ2 could be snugly fitted into the groove, LZ3wCH having the capping helices appeared to cause some steric crash. In order to elaborate this observation, we produced five different deletion mutants and performed Exo I protection assays (Figure 6B). Cleavage of Dmc1-bound ssDNA by Exo I nuclease was suppressed by wild-type Hop2–Mnd1. Importantly, deletion constructs, which retain the LZ3wCH region (Figure 6B; ΔWHD, LZ2+LZ3wCH, LZ3wCH), exhibited a similar level of protection as wild type. In contrast, mutants lacking this region (Figure 6B; WHD+LZ1, ΔLZ3wCH) were far less protective. Thus, contrary to our expectation, the LZ3wCH region alone was sufficient for interacting with the Dmc1 nucleofilament. Induced-fit binding may be necessary for the interaction between LZ3wCH and the groove of the Dmc1 nucleofilament. Consistent with this notion, a proteolysis assay showed that mouse Hop2–Mnd1 complex affects the conformation of human Rad51 (45). Upon binding of LZ3wCH to the Dmc1 nucleofilament, LZ1 and LZ2 may passively position into the helical groove, and the flexibility of the LZ1–LZ2 and LZ2–LZ3 junctions may allow for their adjustments to the induced-fit conformational change of the Dmc1 nucleofilament.

Bottom Line: One end of the rod is linked to two juxtaposed winged-helix domains, and the other end is capped by extra α-helices to form a helical bundle-like structure.Deletion analysis shows that the helical bundle-like structure is sufficient for interacting with the Dmc1-ssDNA nucleofilament, and molecular modeling suggests that the curved rod could be accommodated into the helical groove of the nucleofilament.Remarkably, the winged-helix domains are juxtaposed at fixed relative orientation, and their binding to DNA is likely to perturb the base pairing according to molecular simulations.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, KAIST Institute for the Biocentury, Cancer Metastasis Control Center, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea.

Show MeSH
Related in: MedlinePlus