THE REC2 GENE ENCODES THE HOMOLOGOUS PAIRING PROTEIN OF USTILAGO-MAYDIS

Amino acid sequence analysis has established that the homologous pairi ng protein of Ustilago maydis, known previously in the literature as r eel, is encoded by REC2, a gene essential for recombinational repair a nd meiosis with regional homology to Escherichia coli RecA. The 70-kDa red protein is most likely a proteolytic degradation product of REC2, which has a predicted mass of 84 kDa but which runs anomalously durin g sodium dodecyl sulfate-gel electrophoresis with an apparent mass of 110 kDa. To facilitate purification of the protein product,the REC2 ge ne was overexpressed from a vector that fused a hexahistidine leader s equence onto the amino terminus, enabling isolation of the REC2 protei n on an immobilized metal affinity column. The purified protein exhibi ts ATP-dependent DNA renaturation and DNA-dependent ATPase activities, which were reactions characteristic of the protein as purified from c ell extracts of U. maydis. Homologous pairing activity was established in an assay that measures recognition via non-Watson-Crick bonds betw een identical DNA strands. A size threshold of about 50 bp was found t o govern pairing between linear duplex molecules and homologous single -stranded circles. Joint molecule formation with duplex DNA well under the size threshold was efficiently catalyzed when one strand of the d uplex was composed of RNA. Linear duplex molecules with hairpin caps a lso formed joint molecules when as few as three RNA residues were pres ent.