Gly-103 in the N-terminal domain of Saccharomyces cerevisiae Rad51 protein is critical for DNA binding

Xiao Ping Zhang, Kyung Im Lee, Jachen A. Solinger, Konstantin Kiianitsa, Wolf Dietrich Heyer

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

Rad51 is a homolog of the bacterial RecA protein and is central for recombination in eukaryotes performing homology search and DNA strand exchange. Rad51 and RecA share a core ATPase domain that is structurally similar to the ATPase domains of helicases and the F1 ATPase. Rad51 has an additional N-terminal domain, whereas RecA protein has an additional C-terminal domain. Here we show that glycine 103 in the N-terminal domain of Saccharomyces cerevisiae Rad51 is important for binding to single-stranded and duplex DNA. The Rad51-G103E mutant protein is deficient in DNA strand exchange and ATPase activity due to a primary DNA binding defect. The N-terminal domain of Rad51 is connected to the ATPase core through an extended elbow linker that ensures flexibility of the N-terminal domain. Molecular modeling of the Rad51-G103E mutant protein shows that the negatively charged glutamate residue lies on the surface of the N-terminal domain facing a positively charged patch composed of Arg-260, His-302, and Lys-305 on the ATPase core domain. A possible structural explanation for the DNA binding defect is that a charge interaction between Glu-103 and the positive patch restricts the flexibility of the N-terminal domain. Rad51-G103E was identified in a screen for Rad51 interaction-deficient mutants and was shown to ablate the Rad54 interaction in two-hybrid assays (Krejci, L., Damborsky, J., Thomsen, B., Duno, M., and Bendixen, C. (2001) Mol. Cell. Biol. 21, 966-976). Surprisingly, we found that the physical interaction of Rad51-G103E with Rad54 was not affected. Our data suggest that the two-hybrid interaction defect was an indirect consequence of the DNA binding defect.

Original languageEnglish (US)
Pages (from-to)26303-26311
Number of pages9
JournalJournal of Biological Chemistry
Volume280
Issue number28
DOIs
StatePublished - Jul 15 2005

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Saccharomyces cerevisiae Proteins
Yeast
Adenosine Triphosphatases
Rec A Recombinases
DNA
Mutant Proteins
Defects
Proteins
Two-Hybrid System Techniques
Proton-Translocating ATPases
Bacterial Proteins
Single-Stranded DNA
Elbow
Eukaryota
Molecular modeling
Glycine
Genetic Recombination
Saccharomyces cerevisiae
Glutamic Acid
Assays

ASJC Scopus subject areas

  • Biochemistry

Cite this

Gly-103 in the N-terminal domain of Saccharomyces cerevisiae Rad51 protein is critical for DNA binding. / Zhang, Xiao Ping; Lee, Kyung Im; Solinger, Jachen A.; Kiianitsa, Konstantin; Heyer, Wolf Dietrich.

In: Journal of Biological Chemistry, Vol. 280, No. 28, 15.07.2005, p. 26303-26311.

Research output: Contribution to journalArticle

Zhang, Xiao Ping ; Lee, Kyung Im ; Solinger, Jachen A. ; Kiianitsa, Konstantin ; Heyer, Wolf Dietrich. / Gly-103 in the N-terminal domain of Saccharomyces cerevisiae Rad51 protein is critical for DNA binding. In: Journal of Biological Chemistry. 2005 ; Vol. 280, No. 28. pp. 26303-26311.
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abstract = "Rad51 is a homolog of the bacterial RecA protein and is central for recombination in eukaryotes performing homology search and DNA strand exchange. Rad51 and RecA share a core ATPase domain that is structurally similar to the ATPase domains of helicases and the F1 ATPase. Rad51 has an additional N-terminal domain, whereas RecA protein has an additional C-terminal domain. Here we show that glycine 103 in the N-terminal domain of Saccharomyces cerevisiae Rad51 is important for binding to single-stranded and duplex DNA. The Rad51-G103E mutant protein is deficient in DNA strand exchange and ATPase activity due to a primary DNA binding defect. The N-terminal domain of Rad51 is connected to the ATPase core through an extended elbow linker that ensures flexibility of the N-terminal domain. Molecular modeling of the Rad51-G103E mutant protein shows that the negatively charged glutamate residue lies on the surface of the N-terminal domain facing a positively charged patch composed of Arg-260, His-302, and Lys-305 on the ATPase core domain. A possible structural explanation for the DNA binding defect is that a charge interaction between Glu-103 and the positive patch restricts the flexibility of the N-terminal domain. Rad51-G103E was identified in a screen for Rad51 interaction-deficient mutants and was shown to ablate the Rad54 interaction in two-hybrid assays (Krejci, L., Damborsky, J., Thomsen, B., Duno, M., and Bendixen, C. (2001) Mol. Cell. Biol. 21, 966-976). Surprisingly, we found that the physical interaction of Rad51-G103E with Rad54 was not affected. Our data suggest that the two-hybrid interaction defect was an indirect consequence of the DNA binding defect.",
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T1 - Gly-103 in the N-terminal domain of Saccharomyces cerevisiae Rad51 protein is critical for DNA binding

AU - Zhang, Xiao Ping

AU - Lee, Kyung Im

AU - Solinger, Jachen A.

AU - Kiianitsa, Konstantin

AU - Heyer, Wolf Dietrich

PY - 2005/7/15

Y1 - 2005/7/15

N2 - Rad51 is a homolog of the bacterial RecA protein and is central for recombination in eukaryotes performing homology search and DNA strand exchange. Rad51 and RecA share a core ATPase domain that is structurally similar to the ATPase domains of helicases and the F1 ATPase. Rad51 has an additional N-terminal domain, whereas RecA protein has an additional C-terminal domain. Here we show that glycine 103 in the N-terminal domain of Saccharomyces cerevisiae Rad51 is important for binding to single-stranded and duplex DNA. The Rad51-G103E mutant protein is deficient in DNA strand exchange and ATPase activity due to a primary DNA binding defect. The N-terminal domain of Rad51 is connected to the ATPase core through an extended elbow linker that ensures flexibility of the N-terminal domain. Molecular modeling of the Rad51-G103E mutant protein shows that the negatively charged glutamate residue lies on the surface of the N-terminal domain facing a positively charged patch composed of Arg-260, His-302, and Lys-305 on the ATPase core domain. A possible structural explanation for the DNA binding defect is that a charge interaction between Glu-103 and the positive patch restricts the flexibility of the N-terminal domain. Rad51-G103E was identified in a screen for Rad51 interaction-deficient mutants and was shown to ablate the Rad54 interaction in two-hybrid assays (Krejci, L., Damborsky, J., Thomsen, B., Duno, M., and Bendixen, C. (2001) Mol. Cell. Biol. 21, 966-976). Surprisingly, we found that the physical interaction of Rad51-G103E with Rad54 was not affected. Our data suggest that the two-hybrid interaction defect was an indirect consequence of the DNA binding defect.

AB - Rad51 is a homolog of the bacterial RecA protein and is central for recombination in eukaryotes performing homology search and DNA strand exchange. Rad51 and RecA share a core ATPase domain that is structurally similar to the ATPase domains of helicases and the F1 ATPase. Rad51 has an additional N-terminal domain, whereas RecA protein has an additional C-terminal domain. Here we show that glycine 103 in the N-terminal domain of Saccharomyces cerevisiae Rad51 is important for binding to single-stranded and duplex DNA. The Rad51-G103E mutant protein is deficient in DNA strand exchange and ATPase activity due to a primary DNA binding defect. The N-terminal domain of Rad51 is connected to the ATPase core through an extended elbow linker that ensures flexibility of the N-terminal domain. Molecular modeling of the Rad51-G103E mutant protein shows that the negatively charged glutamate residue lies on the surface of the N-terminal domain facing a positively charged patch composed of Arg-260, His-302, and Lys-305 on the ATPase core domain. A possible structural explanation for the DNA binding defect is that a charge interaction between Glu-103 and the positive patch restricts the flexibility of the N-terminal domain. Rad51-G103E was identified in a screen for Rad51 interaction-deficient mutants and was shown to ablate the Rad54 interaction in two-hybrid assays (Krejci, L., Damborsky, J., Thomsen, B., Duno, M., and Bendixen, C. (2001) Mol. Cell. Biol. 21, 966-976). Surprisingly, we found that the physical interaction of Rad51-G103E with Rad54 was not affected. Our data suggest that the two-hybrid interaction defect was an indirect consequence of the DNA binding defect.

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