Identification of residues of T4 RNase H required for catalysis and DNA binding.
Bacteriophage T4 RNase H, which removes the RNA primers that initiate lagging strand fragments, has a 5'- to 3'-exonuclease activity on DNA.DNA and RNA.DNA duplexes and an endonuclease activity on flap or forked DNA structures (Bhagwat, M., Hobbs, L. J., and Nossal, N. J. (1997) J. Biol. Chem. 272, 28523-28530). It is a member of the RAD2 family of prokaryotic and eukaryotic replication and repair nucleases. The crystal structure of T4 RNase H, in the absence of DNA, shows two Mg2+ ions coordinated to the amino acids highly conserved in this family. It also shows a disordered region proposed to be involved in DNA binding (Mueser, T. C., Nossal, N. G., and Hyde, C. C. Cell (1996) 85, 1101-1112). To identify the amino acids essential for catalysis and DNA binding, we have constructed and characterized three kinds of T4 RNase H mutant proteins based on the possible roles of the amino acid residues: mutants of acidic residues coordinated to each of the two Mg2+ ions (Mg2+-1: D19N, D71N, D132N, and D155N; and Mg2+-2: D157N and D200N); mutants of conserved basic residues in or near the disordered region (K87A and R90A); and mutants of residues with hydroxyl side chains involved in the hydrogen bonding network (Y86F and S153A). Our studies show that Mg2+-1 and the residues surrounding it are important for catalysis and that Lys87 is necessary for DNA binding.
Bhagwat
M
M
Laboratory of Molecular and Cellular Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-0830, USA.
Meara
D
D
Nossal
N G
NG
D000595:Amino Acid Sequence; D017122:Bacteriophage T4 / Q000201:enzymology*; D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D004279:DNA, Viral / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000378:metabolism; D016914:Ribonuclease H / Q000378:metabolism*
D004279:DNA, Viral; D012367:RNA, Viral; D016914:Ribonuclease H; D008274:Magnesium