title: Functional Relationship of ATP Hydrolysis, Presynaptic Filament Stability, and Homologous DNA Pairing Activity of the Human Meiotic Recombinase DMC1.
abstract: DMC1 and RAD51 are conserved recombinases that catalyze homologous recombination. DMC1 and RAD51 share similar properties in DNA binding, DNA-stimulated ATP hydrolysis, and catalysis of homologous DNA strand exchange. A large body of evidence indicates that attenuation of ATP hydrolysis leads to stabilization of the RAD51-ssDNA presynaptic filament and enhancement of DNA strand exchange. However, the functional relationship of ATPase activity, presynaptic filament stability, and DMC1-mediated homologous DNA strand exchange has remained largely unexplored. To address this important question, we have constructed several mutant variants of human DMC1 and characterized them biochemically to gain mechanistic insights. Two mutations, K132R and D223N, that change key residues in the Walker A and B nucleotide-binding motifs ablate ATP binding and render DMC1 inactive. On the other hand, the nucleotide-binding cap D317K mutant binds ATP normally but shows significantly attenuated ATPase activity and, accordingly, forms a highly stable presynaptic filament. Surprisingly, unlike RAD51, presynaptic filament stabilization achieved via ATP hydrolysis attenuation does not lead to any enhancement of DMC1-catalyzed homologous DNA pairing and strand exchange. This conclusion is further supported by examining wild-type DMC1 with non-hydrolyzable ATP analogues. Thus, our results reveal an important mechanistic difference between RAD51 and DMC1.
authors.affiliation: From the Institute of Biochemical Sciences, National Taiwan University, Number 1, Section 4, Roosevelt Road, Taipei 10617 Taiwan.
authors.forename: Hao-Yen
authors.initials: HY
authors.lastname: Chang
authors.affiliation: From the Institute of Biochemical Sciences, National Taiwan University, Number 1, Section 4, Roosevelt Road, Taipei 10617 Taiwan.
authors.forename: Chia-Yu
authors.initials: CY
authors.lastname: Liao
authors.affiliation: From the Institute of Biochemical Sciences, National Taiwan University, Number 1, Section 4, Roosevelt Road, Taipei 10617 Taiwan.
authors.forename: Guan-Chin
authors.initials: GC
authors.lastname: Su
authors.affiliation: the Institute of Biological Chemistry, Academia Sinica, 128 Academia Road, Section 2, Nankang, Taipei 11529, Taiwan, and.
authors.forename: Sheng-Wei
authors.initials: SW
authors.lastname: Lin
authors.affiliation: the Ministry of Education Key Laboratory of Protein Science, Tsinghua-Peking Joint Center for Life Sciences, Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China.
authors.forename: Hong-Wei
authors.initials: HW
authors.lastname: Wang
authors.affiliation: From the Institute of Biochemical Sciences, National Taiwan University, Number 1, Section 4, Roosevelt Road, Taipei 10617 Taiwan, the Institute of Biological Chemistry, Academia Sinica, 128 Academia Road, Section 2, Nankang, Taipei 11529, Taiwan, and peterhchi@ntu.edu.tw.
authors.forename: Peter
authors.initials: P
authors.lastname: Chi
mesh_terms: D000249:Adenosine Monophosphate / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D023902:Chromosome Pairing; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000378:metabolism; D015870:Gene Expression; D059765:Homologous Recombination; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D059372:Nucleotide Motifs; D011485:Protein Binding; D051135:Rad51 Recombinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D018797:Cell Cycle Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D051135:Rad51 Recombinase; D005090:Exodeoxyribonucleases; C489467:RecJ protein, E coli; C074556:DMC1 protein, human
keywords: ATP; ATPase; DMC1; DNA enzyme; DNA recombination; DNA-binding protein; homologous recombination; presynaptic filament; protein-DNA interaction; recombinase activity