Abstract
Bacteriophage T4 RNase H, a flap endonuclease-1 family nuclease, removes RNA primers from lagging strand fragments. It has both 5' nuclease and flap endonuclease activities. Our previous structure of native T4 RNase H (PDB code 1TFR) revealed an active site composed of highly conserved Asp residues and two bound hydrated magnesium ions. Here, we report the crystal structure of T4 RNase H in complex with a fork DNA substrate bound in its active site. This is the first structure of a flap endonuclease-1 family protein with its complete branched substrate. The fork duplex interacts with an extended loop of the helix-hairpin-helix motif class 2. The 5' arm crosses over the active site, extending below the bridge (helical arch) region. Cleavage assays of this DNA substrate identify a primary cut site 7-bases in from the 5' arm. The scissile phosphate, the first bond in the duplex DNA adjacent to the 5' arm, lies above a magnesium binding site. The less ordered 3' arm reaches toward the C and N termini of the enzyme, which are binding sites for T4 32 protein and T4 45 clamp, respectively. In the crystal structure, the scissile bond is located within the double-stranded DNA, between the first two duplex nucleotides next to the 5' arm, and lies above a magnesium binding site. This complex provides important insight into substrate recognition and specificity of the flap endonuclease-1 enzymes.
Highlights
The flap endonuclease-1 (FEN-1)3 nuclease family is conserved in sequence and structure from bacteriophage to humans
Catalytic activity seems opposite to what is usually observed for FEN-1 enzymes, the cleaving specificity of T4 RNase H on these three substrates is the same as that displayed by FEN-1 enzymes [30, 40]
Most structures of FEN-1 enzymes have two divalent metal ions positioned in highly conserved active sites [21, 22, 24, 25, 28]
Summary
Protein Expression and Purification—Recombinant native T4 RNase H (pNN2202) and the inactive D132N mutant (pNN2202-D132N) [29] were expressed in E. coli BL21(DE3) pLysS. Nuclease Assays—For nuclease assays (described in Gangisetty et al [9]), the reaction mixtures of 5 l contained 2 nM 5Ј 32P DNAs and varying concentrations of wild type T4 RNase H, as indicated, B and C. Crystallization of D132N T4 RNase H with Fork DNA Substrate—Purified D132N RNase H was dialyzed at 4 °C against 25 mM Bis-Tris, pH 6.5, 2 mM EDTA, 150 mM NH4Cl, concentrated to 20 mg/ml (Millipore Amicon Ultra, 10,000 molecular weight cutoff), and mixed in an equimolar ratio with fork DNA substrate (10 mg/ml protein). Sparse matrix screening at 23 °C (Emerald BioStructures, Inc.) yielded crystals that were optimized by hanging drop vapor diffusion of droplets composed initially of 2 l of protein, 4 l of water, and 2 l of the well solution The latter consisted of 0.1 M Tris-HCl, pH 7.5, 18% polyethylene glycol 2000 monomethyl ether (MME), and 20% glucose. The Matthews coefficient of 2.7 and the solvent content of 53.4% indicated that the crystallographic asymmetric unit
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