Substrate specificity of the Rad3 ATPase/DNA helicase of Saccharomyces cerevisiae and binding of Rad3 protein to nucleic acids.
Rad3 protein from the yeast Saccharomyces cerevisiae is a single-stranded DNA-dependent ATPase which catalyzes the unwinding of DNA.DNA duplexes. In the present studies we have demonstrated that the purified enzyme additionally catalyzes the displacement of RNA fragments annealed to complementary DNA. Quantitative comparisons using otherwise identical partially duplex DNA.DNA and DNA.RNA substrates indicate a significant preference for the latter. Competition for ATPase or DNA helicase activity by various homopolymers suggests that Rad3 protein does not discriminate between ribonucleotide and deoxyribonucleotide homopolymers with respect to binding. However, neither single-stranded RNA nor various ribonucleotide homopolymers supported the hydrolysis of nucleoside 5'-triphosphates. Additionally, Rad3 protein was unable to catalyze the displacement of oligo(dA) annealed to poly(U), suggesting that the catalytic domain of the enzyme is exquisitely sensitive to chemical and/or or conformational differences between DNA and RNA. Hence, it appears that Rad3 protein is not an RNA helicase.
Naegeli
H
H
Department of Pathology, University of Texas Southwestern Medical Center, Dallas 75235.
Bardwell
L
L
Harosh
I
I
Freidberg
E C
EC
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D001483:Base Sequence; D002384:Catalysis; D004265:DNA Helicases / Q000378:metabolism*; D004271:DNA, Fungal / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D009692:Nucleic Acid Heteroduplexes; D009696:Nucleic Acids / Q000378:metabolism*; D012331:RNA, Fungal / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity
D004271:DNA, Fungal; D009692:Nucleic Acid Heteroduplexes; D009696:Nucleic Acids; D012331:RNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; D000251:Adenosine Triphosphatases; C061897:Rad3 protein, S cerevisiae; D004265:DNA Helicases