DISASSOCIATION OF CONVENTIONAL DNA-BINDING AND ENDONUCLEASE ACTIVITIES BY AN ADENOASSOCIATED VIRUS REP78 MUTANT

As part of Rep78/68's involvement in adeno-associated virus (AAV) DNA replication, these highly related, AAV encoded proteins bind to the AA V terminal repeat (TR) DNA and endonucleolytically cleave one strand a t the terminal resolution site (''trs'' nicking activity) of the TR DN A, a site adjacent to the DNA binding site, 21 bps from the nearest GC TC motif. We have constructed a Rep78 mutant, replacing leucine-threon ine at amino acids 64-65 with histidine-methionine (64(LH)65(TM)). Thi s mutant, expressed as a chimeric protein with maltose binding protein (MBP), displays Mg++ dependent endonuclease activity, but does not bi nd to the AAV TR as determined in the conventional electrophoretic mob ility shift assay (EMSA). It is also found that wild type MBP-Rep78 en donucleolytically nicks at multiple sites in addition to the previousl y recognized trs site. These data suggest that the nicking activity is independent of conventional DNA binding activity as measured by EMSA and further suggest that a separate form of DNA recognition by Rep78, not measured by the EMSA, is taking place which allows for endonucleas e activity. (C) 1995 Academic Press, Inc.