The X-ray structures of 2 complexes of bovine pancreatic deoxyribonuclease (DNase) 1 with octamer duplexes have been refined and showed that binding involves contacts in the minor groove and phosphates from both strands of the deoxyribonucleic acid (DNA). In both cases the DNA is bent away from the enzyme towards the major groove leading to a widening of the minor groove. Together with biochemical results on curved DNA, these structures lead us to conclude that minor groove width and depths and bendability are important helix parameters determining the cutting rates of DNase 1. Cocrystallization of DNase 1 with a methylated oligoribonucleotide in the presence of manganese ions allowed the study of metal binding at the active site.
Efforts to crystallize the Escherichia coli Eco RV methylase in free form as well as in the presence of its cofactor S-adenosyl methionine (SAM) or oligonucleotides have not been successful so far.
The gene for bovine pancreatic DNase 1 has been synthesised chemically and the protein expressed in E. coli. A variety of tightly controlled expression vectors based on the tac, lambda, PL and T7 promoters have been explored. A number of mutants, based on the above crystallographic structures, have been prepared. These have been expressed and their properties studied in order to gain information about particular amino acids.
Kinetic experiments have been carried out with the Eco RV methylase in order to define its mechanism of action. A variety of based analogues have been used to determine the parts of the GATATC sequence that are important in binding to the methylase. This has shown that the major groove area of the first 3 GAT bases are most important in generating selectivity.
Attempts were made to clone and sequence genomic DNA for human DNase 1. Eventually, success was achieved using strain LE392 as the host strain and using large probes derived by polymerase chain reaction (PCR) of human genomic DNA with 2 sets of primers based on the sequence of human complementary DNA (cDNA), one probe of 500 base pairs (bp) from the 5' end of the expressed region and the other of 200 bp from the 3' end.
The enzyme DNA'ase I and the Eco RV methylase will be used to study aspects of protein nucleic acid interactions. The project will involve X-ray crystallography and solution studies such as site-directed mutagenesis, NMR spectroscopy and enzomology. Addtionally DNA'ase I will be cloned from a number of mammals/tissues giving information about strongly conserved parts of the enzyme and its genetic organisation.
Funding SchemeCSC - Cost-sharing contracts