Tyrosine phosphorylation stimulates activity of human RAD51 recombinase through altered nucleoprotein filament dynamics.
The DNA strand exchange protein RAD51 facilitates the central step in homologous recombination, a process fundamentally important for accurate repair of damaged chromosomes, restart of collapsed replication forks, and telomere maintenance. The active form of RAD51 is a nucleoprotein filament that assembles on single-stranded DNA (ssDNA) at the sites of DNA damage. The c-Abl tyrosine kinase and its oncogenic counterpart BCR-ABL fusion kinase phosphorylate human RAD51 on tyrosine residues 54 and 315. We combined biochemical reconstitutions of the DNA strand exchange reactions with total internal reflection fluorescence microscopy to determine how the two phosphorylation events affect the biochemical activities of human RAD51 and properties of the RAD51 nucleoprotein filament. By mimicking RAD51 tyrosine phosphorylation with a nonnatural amino acid, p-carboxymethyl-l-phenylalanine (pCMF), we demonstrated that Y54 phosphorylation enhances the RAD51 recombinase activity by at least two different mechanisms, modifies the RAD51 nucleoprotein filament formation, and allows RAD51 to compete efficiently with ssDNA binding protein RPA. In contrast, Y315 phosphorylation has little effect on the RAD51 activities. Based on our work and previous cellular studies, we propose a mechanism underlying RAD51 activation by c-Abl/BCR-ABL kinases.
Department of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City, IA 52242; Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801.
Shyamal
S
Subramanyam
Department of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City, IA 52242.
Mohammed
M
Ismail
Department of Electrical and Computer Engineering, University of Iowa, Iowa City, IA 52242.
Ipshita
I
Bhattacharya
Department of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City, IA 52242; maria-spies@uiowa.edu.
Maria
0000-0002-7375-8037
M
Spies
D000067558:Biological Mimicry; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004789:Enzyme Activation; D059765:Homologous Recombination; D006801:Humans; D006868:Hydrolysis; D008958:Models, Molecular; D009154:Mutation; D009698:Nucleoproteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D055503:Protein Multimerization; D016315:Proto-Oncogene Proteins c-abl / Q000378:metabolism; D051135:Rad51 Recombinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009698:Nucleoproteins; D011994:Recombinant Proteins; D019000:Phosphotyrosine; D004247:DNA; D016315:Proto-Oncogene Proteins c-abl; D051135:Rad51 Recombinase
RAD51 recombinase; c-Abl tyrosine kinase; homologous recombination; phosphorylation; single-molecule total internal reflection fluorescence microscopy
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