Tyrosine phosphorylation stimulates activity of human RAD51 recombinase through altered nucleoprotein filament dynamics

Shyamal Subramanyam; Mohammed Ismail; Ipshita Bhattacharya; Maria Spies

doi:10.1073/pnas.1604807113

Back

Tyrosine phosphorylation stimulates activity of human RAD51 recombinase through altered nucleoprotein filament dynamics

Journal article

Open access

Peer reviewed

Tyrosine phosphorylation stimulates activity of human RAD51 recombinase through altered nucleoprotein filament dynamics

Shyamal Subramanyam, Mohammed Ismail, Ipshita Bhattacharya and Maria Spies

Proceedings of the National Academy of Sciences - PNAS, Vol.113(41), pp.E6045-E6054

10/11/2016

DOI: 10.1073/pnas.1604807113

PMCID: PMC5068273

PMID: 27671650

Files and links (1)

url

https://doi.org/10.1073/pnas.1604807113View

Published (Version of record) Open Access

Abstract

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.

Nucleoproteins - chemistry

Rad51 Recombinase - metabolism

Phosphorylation

Rad51 Recombinase - genetics

DNA, Single-Stranded - metabolism

Humans

Protein Multimerization

Models, Molecular

Homologous Recombination

DNA - metabolism

Recombinant Proteins

Biological Mimicry

Phosphotyrosine - chemistry

Phosphotyrosine - metabolism

DNA - genetics

DNA-Binding Proteins - metabolism

Hydrolysis

Nucleoproteins - metabolism

Rad51 Recombinase - chemistry

Proto-Oncogene Proteins c-abl - metabolism

Protein Conformation

Enzyme Activation

Mutation

Phosphotyrosine - genetics

Details

Title: Subtitle: Tyrosine phosphorylation stimulates activity of human RAD51 recombinase through altered nucleoprotein filament dynamics
Creators: Shyamal Subramanyam - 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
Mohammed Ismail - Department of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City, IA 52242
Ipshita Bhattacharya - Department of Electrical and Computer Engineering, University of Iowa, Iowa City, IA 52242
Maria Spies - Department of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City, IA 52242; maria-spies@uiowa.edu
Resource Type: Journal article
Publication Details: Proceedings of the National Academy of Sciences - PNAS, Vol.113(41), pp.E6045-E6054
DOI: 10.1073/pnas.1604807113
PMID: 27671650
PMCID: PMC5068273
NLM abbreviation: Proc Natl Acad Sci U S A
ISSN: 0027-8424
eISSN: 1091-6490
Publisher: National Academy of Sciences; United States
Grant note: R01 GM108617 / NIGMS NIH HHS P30 CA086862 / NCI NIH HHS
Language: English
Date published: 10/11/2016
Academic Unit: Electrical and Computer Engineering; Radiation Oncology; Biochemistry and Molecular Biology
Record Identifier: 9984024551402771

Metrics

17 Record Views

48 Times Cited - Web of Science