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 (hDMC1K132R) or alanine (hDMC1K132A). The hDMC1 variants were expressed in Escherichia coli and purified to near homogeneity. Both hDMC1K132R and hDMC1K132A variants were devoid of ATP hydrolysis. The hDMC1K132R variant was attenuated for ATP binding that was partially restored by the addition of either ssDNA or calcium. The hDMC1K132R variant was partially capable of homologous DNA pairing and strand exchange in the presence of calcium and protecting DNA from a nuclease, while the hDMC1K132A 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.

Functional Relationship of ATP Hydrolysis, Presynaptic Filament Stability, and Homologous DNA Pairing Activity of the Human Meiotic Recombinase DMC1

“…DMC1 also requires ATP for the assembly of a functional presynaptic filament and to perform homologous DNA pairing and strand exchange. However, studies that have been conducted thus far provide no evidence for an up-regulation of DMC1 recombinase activity upon attenuation of ATP hydrolysis by the presynaptic filament (22,33,34). These observations suggest important mechanistic differences between RAD51 and DMC1 with regards to presynaptic filament assembly and recombinase activity.…”

“…We note that neither the K132R nor the D223N variant is capable of ATP binding. Accordingly, these mutants also lack the ability to form a functional presynaptic filament as revealed by their failure to protect ssDNA against Exonuclease I challenge or to perform DNA strand exchange (34) (Fig. 8).…”

“…DNA Binding Properties of the DMC1 D317K Mutant-It has been documented that DMC1-mediated ATP hydrolysis is stimulated by DNA (20,34), so it is important to determine whether the reduced activity of DMC1 D317K in ATP hydrolysis might stem from a defect in DNA binding, We used an electrophoretic mobility shift assay to analyze the DNA binding 5 and 9). C, DMC1 (WT) and DMC1 D317K (lanes 2 and 6, 0.5 M; lanes 3 and 7, 1 M; lanes 4,5,8, and 9, 3 M) were incubated with 32 P-labeled 80-mer ssDNA (3 M nucleotides).…”

Characterization of the recombination activities of the Entamoeba histolytica Rad51 recombinase

“…To test the formation of a presynaptic filament, we utilized a nuclease protection assay [15,37,45,52]. In this assay, eh Rad51 is incubated with 32 P-labeled ssDNA in the presence of ATP to allow for filament formation, followed by the addition of DNase I to degrade any exposed DNA.…”

“…OL90 (ssDNA) was used in the nuclease protection assay and D-loop assay. All oligonucleotides indicated as radiolabeled were done so using T4 polynucleotide kinase and [ 32 P-γ]-ATP as described [15]. ϕX174 (+) virion ssDNA and ϕX174 replicative form I dsDNA were purchased from New England BioLabs; the ϕX174 replicative form I dsDNA was linearized with Apa LI (New England BioLabs).…”