Interactions of Human Rad54 Protein with Branched DNA Molecules

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

doi:10.1074/jbc.m701992200

Olga M Mazina, Matthew J Rossi + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m701992200

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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, approximately 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.

Highlights

Several mechanisms were proposed to account for this stimulation
HRad54 Shows Binding Preferences for Branched DNA Substrates with ssDNA Arms—Previously, we found that human Rad54 (hRad54) shows DNA binding preference for branched DNA substrates [25]. hRad54 possesses a dsDNA-dependent ATPase activity, which is essential for DNA branch migration and DNA translocation
We measured the initial rate of ATP hydrolysis of hRad54 protein in the presence of various branched oligonucleotide-derived DNA substrates over a range of DNA concentrations (Fig. 1)

Summary

EXPERIMENTAL PROCEDURES

Proteins—GST- and His6-tagged versions of hRad protein were expressed in Sf21 insect cells [11]. The reactions in standard buffer containing 25 mM Tris acetate, pH 7.5, 10 mM magnesium acetate (unless indicated otherwise), 1 mM dithiothreitol, 2 mM ATP, 3 mM phosphoenolpyruvate, pyruvate kinase (20 units/ml), lactate dehydrogenase (20 units/ ml), NADH (200 ␮g/ml), and the indicated concentrations of Rad protein and DNA were carried out at 30 °C. Branch Migration Assay—The hRad protein (100 nM, unless indicated otherwise) was incubated with 32P-labeled synthetic PX-junction (number 71/169/170/171) (33 nM, molecules), 32P-labeled synthetic X-junction (number 71/170/234/ 235) (33 nM, molecules), or 32P-labeled synthetic PX-junction (number 265/266/269/270) (20 or 30 nM, molecules) in a 90-␮l branch migration buffer containing 25 mM Tris acetate, pH 7.5, 2 mM ATP, 1 mM dithiothreitol, 100 ␮g/ml bovine serum albumin, the ATP-regenerating system (10 units/ml creatine phosphokinase and 15 mM creatine phosphate), and the indicated concentrations of magnesium acetate. Creatine phosphate, with no apparent effect on the initial rate measurements

RESULTS

Oligonucleotidesa bp

DISCUSSION