Interactions of human Rad54 protein with branched DNA molecules

Olga M. Mazina, Matthew J. Rossi, Nicolas H. Thomä, Alexander V. Mazin

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

The Rad54 protein plays an important role during homologous recombination in eukaryotes. The protein belongs to the Swi2/Snf2 family of ATP-dependent DNA translocases. We previously showed that yeast and human Rad54 (hRad54) specifically bind to Holliday junctions and promote branch migration. Here we examined the minimal DNA structural requirements for optimal hRad54 ATPase and branch migration activity. Although a 12-bp double-stranded DNA region of branched DNA is sufficient to induce ATPase activity, the minimal substrate that gave rise to optimal stimulation of the ATP hydrolysis rate consisted of two short double-stranded DNA arms, 15 bp each, combined with a 45-nucleotide single-stranded DNA branch. We showed that hRad54 binds preferentially to the open and not to the stacked conformation of branched DNA. Stoichiometric titration of hRad54 revealed formation of two types of hRad54 complexes with branched DNA substrates. The first of them, a dimer, is responsible for the ATPase activity of the protein. However, branch migration activity requires a significantly higher stoichiometry of hRad54, ∼10 ± 2 protein monomers/DNA molecule. This pleomorphism of hRad54 in formation of oligomeric complexes with DNA may correspond to multiple functions of the protein in homologous recombination.

Original languageEnglish (US)
Pages (from-to)21068-21080
Number of pages13
JournalJournal of Biological Chemistry
Volume282
Issue number29
DOIs
StatePublished - Jul 20 2007

Fingerprint

Molecules
DNA
Adenosine Triphosphatases
Homologous Recombination
Proteins
Adenosine Triphosphate
Cruciform DNA
Nucleic Acid Conformation
human RAD54L protein
Single-Stranded DNA
Eukaryota
Substrates
Titration
Stoichiometry
Dimers
Yeast
Hydrolysis
Nucleotides
Conformations
Yeasts

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Mazina, Olga M. ; Rossi, Matthew J. ; Thomä, Nicolas H. ; Mazin, Alexander V. / Interactions of human Rad54 protein with branched DNA molecules. In: Journal of Biological Chemistry. 2007 ; Vol. 282, No. 29. pp. 21068-21080.
@article{8ac513ddd55b4141a21ef8d26bc47518,
title = "Interactions of human Rad54 protein with branched DNA molecules",
abstract = "The Rad54 protein plays an important role during homologous recombination in eukaryotes. The protein belongs to the Swi2/Snf2 family of ATP-dependent DNA translocases. We previously showed that yeast and human Rad54 (hRad54) specifically bind to Holliday junctions and promote branch migration. Here we examined the minimal DNA structural requirements for optimal hRad54 ATPase and branch migration activity. Although a 12-bp double-stranded DNA region of branched DNA is sufficient to induce ATPase activity, the minimal substrate that gave rise to optimal stimulation of the ATP hydrolysis rate consisted of two short double-stranded DNA arms, 15 bp each, combined with a 45-nucleotide single-stranded DNA branch. We showed that hRad54 binds preferentially to the open and not to the stacked conformation of branched DNA. Stoichiometric titration of hRad54 revealed formation of two types of hRad54 complexes with branched DNA substrates. The first of them, a dimer, is responsible for the ATPase activity of the protein. However, branch migration activity requires a significantly higher stoichiometry of hRad54, ∼10 ± 2 protein monomers/DNA molecule. This pleomorphism of hRad54 in formation of oligomeric complexes with DNA may correspond to multiple functions of the protein in homologous recombination.",
author = "Mazina, {Olga M.} and Rossi, {Matthew J.} and Thom{\"a}, {Nicolas H.} and Mazin, {Alexander V.}",
year = "2007",
month = "7",
day = "20",
doi = "10.1074/jbc.M701992200",
language = "English (US)",
volume = "282",
pages = "21068--21080",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "29",

}

Interactions of human Rad54 protein with branched DNA molecules. / Mazina, Olga M.; Rossi, Matthew J.; Thomä, Nicolas H.; Mazin, Alexander V.

In: Journal of Biological Chemistry, Vol. 282, No. 29, 20.07.2007, p. 21068-21080.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Interactions of human Rad54 protein with branched DNA molecules

AU - Mazina, Olga M.

AU - Rossi, Matthew J.

AU - Thomä, Nicolas H.

AU - Mazin, Alexander V.

PY - 2007/7/20

Y1 - 2007/7/20

N2 - The Rad54 protein plays an important role during homologous recombination in eukaryotes. The protein belongs to the Swi2/Snf2 family of ATP-dependent DNA translocases. We previously showed that yeast and human Rad54 (hRad54) specifically bind to Holliday junctions and promote branch migration. Here we examined the minimal DNA structural requirements for optimal hRad54 ATPase and branch migration activity. Although a 12-bp double-stranded DNA region of branched DNA is sufficient to induce ATPase activity, the minimal substrate that gave rise to optimal stimulation of the ATP hydrolysis rate consisted of two short double-stranded DNA arms, 15 bp each, combined with a 45-nucleotide single-stranded DNA branch. We showed that hRad54 binds preferentially to the open and not to the stacked conformation of branched DNA. Stoichiometric titration of hRad54 revealed formation of two types of hRad54 complexes with branched DNA substrates. The first of them, a dimer, is responsible for the ATPase activity of the protein. However, branch migration activity requires a significantly higher stoichiometry of hRad54, ∼10 ± 2 protein monomers/DNA molecule. This pleomorphism of hRad54 in formation of oligomeric complexes with DNA may correspond to multiple functions of the protein in homologous recombination.

AB - The Rad54 protein plays an important role during homologous recombination in eukaryotes. The protein belongs to the Swi2/Snf2 family of ATP-dependent DNA translocases. We previously showed that yeast and human Rad54 (hRad54) specifically bind to Holliday junctions and promote branch migration. Here we examined the minimal DNA structural requirements for optimal hRad54 ATPase and branch migration activity. Although a 12-bp double-stranded DNA region of branched DNA is sufficient to induce ATPase activity, the minimal substrate that gave rise to optimal stimulation of the ATP hydrolysis rate consisted of two short double-stranded DNA arms, 15 bp each, combined with a 45-nucleotide single-stranded DNA branch. We showed that hRad54 binds preferentially to the open and not to the stacked conformation of branched DNA. Stoichiometric titration of hRad54 revealed formation of two types of hRad54 complexes with branched DNA substrates. The first of them, a dimer, is responsible for the ATPase activity of the protein. However, branch migration activity requires a significantly higher stoichiometry of hRad54, ∼10 ± 2 protein monomers/DNA molecule. This pleomorphism of hRad54 in formation of oligomeric complexes with DNA may correspond to multiple functions of the protein in homologous recombination.

UR - http://www.scopus.com/inward/record.url?scp=34547136691&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=34547136691&partnerID=8YFLogxK

U2 - 10.1074/jbc.M701992200

DO - 10.1074/jbc.M701992200

M3 - Article

C2 - 17545145

AN - SCOPUS:34547136691

VL - 282

SP - 21068

EP - 21080

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 29

ER -