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 Ust ilago maydis yields a protein preparation that is enriched for a 70-kD a polypeptide as determined by SDS-gel electrophoresis. The protein re sponsible for the ATP-dependent pairing activity, using renaturation o f 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 th e interpretation that the activity arises from a monomeric globular pr otein of 70 kDa. Including heparin-agarose and FPLC gel filtration chr omatography steps in the previously published protocol improves the pu rification of the protein. ATP and Mg2+ are necessary cofactors for op timal DNA renaturation activity. ADP inhibits the reaction. Analysis o f the ATP-dependent renaturation kinetics indicates the reaction proce eds 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 col umn. Single-stranded DNA and Mg2+ are required for optimal ATP hydroly tic activity, although a number of other polynucleotides and divalent cations can substitute to varying degrees. Hydrolysis of ATP is activa ted in a sigmoidal manner with increasing amounts of the protein. At A TP concentrations below 0.1 mM the ATPase activity exhibits positive c ooperativity 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 differe nt modes. These results are interpreted as evidence for protein-protei n interactions.