Abstract
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.
Highlights
The 5Ј- to 3Ј-nuclease of T4 RNase H degrades both RNA1⁄7DNA and DNA1⁄7DNA duplexes, releasing short oligonucleotide products from the 5Ј-end
To determine the amino acids important for the cleavage of the phosphodiester bond by T4 RNase H, we selected residues for mutation based on the crystal structure of T4 RNase H [4] and its sequence similarity to other prokaryotic and eukaryotic nucleases (Fig. 1A)
As a step toward understanding the mechanism of this important enzyme, we have constructed and characterized T4 RNase H mutants altered in active site residues that are coordinated to the two Mg2ϩ, are in the disordered region, or possess hydroxyl side chains
Summary
Vol 272, No 45, Issue of November 7, pp. 28531–28538, 1997 Printed in U.S.A. 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 5to 3-exonuclease activity on DNA1⁄7DNA and RNA1⁄7DNA duplexes and an endonuclease activity on flap or forked DNA structures Like T4 RNase H, many of these enzymes are 5Ј-nucleases that remove primers initiating lagging strand fragments These include the bacteriophage T5 D15 and T7 gene 6 exonucleases, the 5Ј- to 3Ј-exonuclease domains of DNA polymerases from bacteria such as E. coli, Mycobacterium tuberculosis (Mtb), and Thermus aquatics (Taq), and the eukaryotic nucleases such as murine FEN2-1 and human RAD2 analog ( called MF-1 or FEN-1) (for review, see Ref. 4). We report the effects of these mutations on the exonuclease activity, the flap endonuclease activity, and on binding to DNA The implications of these results on the possible roles of the two Mg2ϩ ions and the active site residues are discussed
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