MUTATIONAL ANALYSIS OF AGROBACTERIUM-TUMEFACIENS PTIA6 VIRD1 - IDENTIFICATION OF FUNCTIONALLY IMPORTANT RESIDUES

Mutagenesis experiments were used to identify functionally important r egions of Agrobacterium tumefaciens pTiA6 VirD1. Random mutations were introduced by using Tag polymerase in a mutagenic reaction buffer con taining manganese and altered nucleotide ratios to increase errors dur ing the polymerase chain reaction (PCR). The mutants were assayed for VirD1-, VirD2-dependent border-nicking activity in Escherichia coli ha rbouring a border-containing substrate plasmid. Analysis of the mutant s led to the identification of a region from amino acids 45-60 that is important for VirD1 activity. This region corresponds to a previously postulated potential DNA-binding domain. Deletion mutagenesis indicat ed that amino acids 2-16 could be deleted without affecting VirD1 func tion, whereas a larger deletion, amino acids 5-27, completely inactiva ted VirD1.