ATP-dependent DNA renaturation and DNA-dependent ATPase reactions catalyzed by the Ustilago maydis homologous pairing protein.
Purification of the ATP-dependent homologous pairing activity from Ustilago maydis yields a protein preparation that is enriched for a 70-kDa polypeptide as determined by SDS-gel electrophoresis. The protein responsible for the ATP-dependent pairing activity, using renaturation of complementary single strands of DNA as an assay, has a Stokes radius of 3.6 nm and a sedimentation coefficient of 4.3 S consistent with the interpretation that the activity arises from a monomeric globular protein of 70 kDa. Including heparin-agarose and FPLC gel filtration chromatography steps in the previously published protocol improves the purification of the protein. ATP and Mg2+ are necessary cofactors for optimal DNA renaturation activity. ADP inhibits the reaction. Analysis of the ATP-dependent renaturation kinetics indicates the reaction proceeds through a first-order mechanism. The protein has an associated DNA-dependent ATPase as indicated by co-chromatography with the purified ATP-dependent renaturation activity through an FPLC gel-filtration column. Single-stranded DNA and Mg2+ are required for optimal ATP hydrolytic activity, although a number of other polynucleotides and divalent cations can substitute to varying degrees. Hydrolysis of ATP is activated in a sigmoidal manner with increasing amounts of the protein. At ATP concentrations below 0.1 mM the ATPase activity exhibits positive cooperativity as indicated from the Hill coefficient of 1.8 determined by steady-state kinetic analysis of the reaction. ADP and adenosine 5'-[beta,gamma-imido]triphosphate are inhibitors of the ATPase activity although they appear to exert their inhibitory effects through different modes. These results are interpreted as evidence for protein-protein interactions.
Kmiec
E B
EB
Department of Pharmacology, Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia.
Holloman
W K
WK
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D004265:DNA Helicases; D004271:DNA, Fungal / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D005990:Glycerol; D006868:Hydrolysis; D007700:Kinetics; D009695:Nucleic Acid Renaturation; D011995:Recombination, Genetic; D014588:Ustilago / Q000235:genetics / Q000378:metabolism*
D004271:DNA, Fungal; D005656:Fungal Proteins; D000255:Adenosine Triphosphate; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; D005990:Glycerol