title: Role of the conserved lysine within the Walker A motif of human DMC1.
abstract: During meiosis, the RAD51 recombinase and its meiosis-specific homolog DMC1 mediate DNA strand exchange between homologous chromosomes. The proteins form a right-handed nucleoprotein complex on ssDNA called the presynaptic filament. In an ATP-dependent manner, the presynaptic filament searches for homology to form a physical connection with the homologous chromosome. We constructed two variants of hDMC1 altering the conserved lysine residue of the Walker A motif to arginine (hDMC1(K132R)) or alanine (hDMC1(K132A)). The hDMC1 variants were expressed in Escherichia coli and purified to near homogeneity. Both hDMC1(K132R) and hDMC1(K132A) variants were devoid of ATP hydrolysis. The hDMC1(K132R) variant was attenuated for ATP binding that was partially restored by the addition of either ssDNA or calcium. The hDMC1(K132R) variant was partially capable of homologous DNA pairing and strand exchange in the presence of calcium and protecting DNA from a nuclease, while the hDMC1(K132A) variant was inactive. These results suggest that the conserved lysine of the Walker A motif in hDMC1 plays a key role in ATP binding. Furthermore, the binding of calcium and ssDNA promotes a conformational change in the ATP binding pocket of hDMC1 that promotes ATP binding. Our results provide evidence that the conserved lysine in the Walker A motif of hDMC1 is critical for ATP binding which is required for presynaptic filament formation.
authors.affiliation: Department of Genetics and Biochemistry, Clemson University, Clemson, SC 29634, USA.
authors.forename: Deepti
authors.initials: D
authors.lastname: Sharma
authors.forename: Amanda F
authors.initials: AF
authors.lastname: Say
authors.forename: LeAnna L
authors.initials: LL
authors.lastname: Ledford
authors.forename: Ami J
authors.initials: AJ
authors.lastname: Hughes
authors.forename: Hilarie A
authors.initials: HA
authors.lastname: Sehorn
authors.forename: Donard S
authors.initials: DS
authors.lastname: Dwyer
authors.forename: Michael G
authors.initials: MG
authors.lastname: Sehorn
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000409:Alanine / Q000235:genetics; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001120:Arginine / Q000235:genetics; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D008239:Lysine / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D017434:Protein Structure, Tertiary
chemical_list: D018797:Cell Cycle Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D000255:Adenosine Triphosphate; D001120:Arginine; C074556:DMC1 protein, human; D008239:Lysine; D000409:Alanine; D002118:Calcium