Mutational analysis of Agrobacterium tumefaciens pTiA6 virD1: identification of functionally important residues

Mutagenesis experiments were used to identify functionally important regions of Agrobacterium tumefaciens pTiA6 VirD1. Random mutations were introduced by using Taq polymerase in a mutagenic reaction buffer containing manganese and altered nucleotide ratios to increase errors during the polymerase chain reaction (PCR). The mutants were assayed for VirD1‐, VirD2‐dependent border‐nicking activity in Escherichia coli harbouring a border‐containing substrate plasmid. Analysis of the mutants 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 indicated that amino acids 2–16 could be deleted without affecting VirD1 function, whereas a larger deletion, amino acids 5–27, completely inactivated VirD1.