The N-terminal domain of the human Rad51 protein binds DNA: Structure and a DNA binding surface as revealed by NMR

Hideki Aihara; Yutaka Ito; Hitoshi Kurumizaka; Shigeyuki Yokoyama; Takehiko Shibata

doi:10.1006/jmbi.1999.2904

The N-terminal domain of the human Rad51 protein binds DNA: Structure and a DNA binding surface as revealed by NMR

Hideki Aihara, Yutaka Ito, Hitoshi Kurumizaka, Shigeyuki Yokoyama, Takehiko Shibata

Biochemistry, Molecular Biology, and Biophysics (CBS)

Research output: Contribution to journal › Article › peer-review

120 Scopus citations

Abstract

Human Rad51 protein (HsRad51) is a homolog of Escherichia coli RecA protein, and functions in DNA repair and recombination. In higher eukaryotes, Rad51 protein is essential for cell viability. The N-terminal region of HsRad51 is highly conserved among eukaryotic Rad51 proteins but is absent from RecA, suggesting a Rad51-specific function for this region. Here, we have determined the structure of the N-terminal part of HsRad51 by NMR spectroscopy. The N-terminal region forms a compact domain consisting of five short helices, which shares structural similarity with a domain of endonuclease III, a DNA repair enzyme of E. coli. NMR experiments did not support the involvement of the N-terminal domain in HsRad51-HsBrca2 interaction or the self-association of HsRad51 as proposed by previous studies. However, NMR tiration experiments demonstrated a physical interaction of the domain with DNA, and allowed mapping of the DNA binding surface. Mutation analysis showed that the DNA binding surface is essential for double-stranded and single-stranded DNA binding of HsRad51. Our results suggest the presence of a DNA binding site on the outside surface of the HsRad51 filament and provide a possible explanation for the regulation of DNA binding by phosphorylation within the N-terminal domain.

Original language	English (US)
Pages (from-to)	495-504
Number of pages	10
Journal	Journal of Molecular Biology
Volume	290
Issue number	2
DOIs	https://doi.org/10.1006/jmbi.1999.2904
State	Published - Jul 9 1999

Bibliographical note

Funding Information:
We thank Dr N. Dohmae and Dr K. Takio (Division of Biomolecular Characterization, RIKEN) for mass spectrometry, Dr T. Terada (Division of Biomolecular Characterization, RIKEN; Department of Biophysics and Biochemistry, The Graduate School of Science, The University of Tokyo) for helpful discussions. This work was supported by grants from the Biodesign Research Program, and MR Science Program from RIKEN, a grant from the Ministry of Education, Science and Culture, Japan, a research grant from Human Frontier Science Program (RG493/95), and a grant for CREST from JST (Japan Science and Technology) (Y.I. and T.S.).

Keywords

DNA binding
Genetic recombination
NMR spectroscopy
Rad51 protein
Solution structure

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10.1006/jmbi.1999.2904

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title = "The N-terminal domain of the human Rad51 protein binds DNA: Structure and a DNA binding surface as revealed by NMR",

abstract = "Human Rad51 protein (HsRad51) is a homolog of Escherichia coli RecA protein, and functions in DNA repair and recombination. In higher eukaryotes, Rad51 protein is essential for cell viability. The N-terminal region of HsRad51 is highly conserved among eukaryotic Rad51 proteins but is absent from RecA, suggesting a Rad51-specific function for this region. Here, we have determined the structure of the N-terminal part of HsRad51 by NMR spectroscopy. The N-terminal region forms a compact domain consisting of five short helices, which shares structural similarity with a domain of endonuclease III, a DNA repair enzyme of E. coli. NMR experiments did not support the involvement of the N-terminal domain in HsRad51-HsBrca2 interaction or the self-association of HsRad51 as proposed by previous studies. However, NMR tiration experiments demonstrated a physical interaction of the domain with DNA, and allowed mapping of the DNA binding surface. Mutation analysis showed that the DNA binding surface is essential for double-stranded and single-stranded DNA binding of HsRad51. Our results suggest the presence of a DNA binding site on the outside surface of the HsRad51 filament and provide a possible explanation for the regulation of DNA binding by phosphorylation within the N-terminal domain.",

keywords = "DNA binding, Genetic recombination, NMR spectroscopy, Rad51 protein, Solution structure",

author = "Hideki Aihara and Yutaka Ito and Hitoshi Kurumizaka and Shigeyuki Yokoyama and Takehiko Shibata",

note = "Funding Information: We thank Dr N. Dohmae and Dr K. Takio (Division of Biomolecular Characterization, RIKEN) for mass spectrometry, Dr T. Terada (Division of Biomolecular Characterization, RIKEN; Department of Biophysics and Biochemistry, The Graduate School of Science, The University of Tokyo) for helpful discussions. This work was supported by grants from the Biodesign Research Program, and MR Science Program from RIKEN, a grant from the Ministry of Education, Science and Culture, Japan, a research grant from Human Frontier Science Program (RG493/95), and a grant for CREST from JST (Japan Science and Technology) (Y.I. and T.S.).",

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doi = "10.1006/jmbi.1999.2904",

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T1 - The N-terminal domain of the human Rad51 protein binds DNA

T2 - Structure and a DNA binding surface as revealed by NMR

AU - Aihara, Hideki

AU - Ito, Yutaka

AU - Kurumizaka, Hitoshi

AU - Yokoyama, Shigeyuki

AU - Shibata, Takehiko

N1 - Funding Information: We thank Dr N. Dohmae and Dr K. Takio (Division of Biomolecular Characterization, RIKEN) for mass spectrometry, Dr T. Terada (Division of Biomolecular Characterization, RIKEN; Department of Biophysics and Biochemistry, The Graduate School of Science, The University of Tokyo) for helpful discussions. This work was supported by grants from the Biodesign Research Program, and MR Science Program from RIKEN, a grant from the Ministry of Education, Science and Culture, Japan, a research grant from Human Frontier Science Program (RG493/95), and a grant for CREST from JST (Japan Science and Technology) (Y.I. and T.S.).

PY - 1999/7/9

Y1 - 1999/7/9

N2 - Human Rad51 protein (HsRad51) is a homolog of Escherichia coli RecA protein, and functions in DNA repair and recombination. In higher eukaryotes, Rad51 protein is essential for cell viability. The N-terminal region of HsRad51 is highly conserved among eukaryotic Rad51 proteins but is absent from RecA, suggesting a Rad51-specific function for this region. Here, we have determined the structure of the N-terminal part of HsRad51 by NMR spectroscopy. The N-terminal region forms a compact domain consisting of five short helices, which shares structural similarity with a domain of endonuclease III, a DNA repair enzyme of E. coli. NMR experiments did not support the involvement of the N-terminal domain in HsRad51-HsBrca2 interaction or the self-association of HsRad51 as proposed by previous studies. However, NMR tiration experiments demonstrated a physical interaction of the domain with DNA, and allowed mapping of the DNA binding surface. Mutation analysis showed that the DNA binding surface is essential for double-stranded and single-stranded DNA binding of HsRad51. Our results suggest the presence of a DNA binding site on the outside surface of the HsRad51 filament and provide a possible explanation for the regulation of DNA binding by phosphorylation within the N-terminal domain.

AB - Human Rad51 protein (HsRad51) is a homolog of Escherichia coli RecA protein, and functions in DNA repair and recombination. In higher eukaryotes, Rad51 protein is essential for cell viability. The N-terminal region of HsRad51 is highly conserved among eukaryotic Rad51 proteins but is absent from RecA, suggesting a Rad51-specific function for this region. Here, we have determined the structure of the N-terminal part of HsRad51 by NMR spectroscopy. The N-terminal region forms a compact domain consisting of five short helices, which shares structural similarity with a domain of endonuclease III, a DNA repair enzyme of E. coli. NMR experiments did not support the involvement of the N-terminal domain in HsRad51-HsBrca2 interaction or the self-association of HsRad51 as proposed by previous studies. However, NMR tiration experiments demonstrated a physical interaction of the domain with DNA, and allowed mapping of the DNA binding surface. Mutation analysis showed that the DNA binding surface is essential for double-stranded and single-stranded DNA binding of HsRad51. Our results suggest the presence of a DNA binding site on the outside surface of the HsRad51 filament and provide a possible explanation for the regulation of DNA binding by phosphorylation within the N-terminal domain.

KW - DNA binding

KW - Genetic recombination

KW - NMR spectroscopy

KW - Rad51 protein

KW - Solution structure

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JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

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ER -

The N-terminal domain of the human Rad51 protein binds DNA: Structure and a DNA binding surface as revealed by NMR

Abstract

Bibliographical note

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