Bacteriophage T4 RNase H is a 5' to 3' exonuclease that removes RNA primers from the lagging strand of the DNA replication fork and is a member of the RAD2 family of eukaryotic and prokaryotic replication and repair nucleases. The crystal structure of the full-length native form of T4 RNase H has been solved at 2.06 angstroms resolution in the presence of Mg2+ but in the absence of nucleic acids. The most conserved residues are clustered together in a large cleft with two Mg2+ in the proposed active site. This structure suggests the way in which the widely separated conserved regions in the larger nucleotide excision repair proteins, such as human XPG, could assemble into a structure like that of the smaller replication nucleases.

Bacteriophage T4 DNA polymerase has a proofreading 3' -+ 5' exonuclease that plays an important role in maintaining the accuracy of DNA replication. We have constructed a T4 DNA polymerase deficient in this exonuclease by converting Asp-219 to Ala. The exonuclease activity of the mutant T4 DNA polymerase has been reduced by a factor of at least 107, but it retains a polymerase activity whose kinetic parameters, k at, Kd DNA, and Kd dATP, are very (17-20). We wished to construct a mutation in T4 DNA polymerase that would remove this exonuclease activity, without altering the polymerase activity, in order to evaluate the role of the exonuclease in maintaining the fidelity of DNA replication and to facilitate kinetic studies of the mechanism by which T4 DNA polymerase alone catalyzes DNA synthesis. Previous studies from one of our laboratories (21) to define a minimal kinetic scheme for T4 DNA polymerase (Scheme I) were complicated by the vigorous exonuclease activity of this enzyme. Exonucleasedeficient enzymes of T7 DNA polymerase (22, 23) and E. coli DNA polymerase I (Klenow fragment) (24) have played integral roles in the delineation of their kinetic mechanisms.The active site for exonuclease must be toward the N terminus ofT4 polymerase, since the protein made by the B22 amber mutant, missing the C-terminal 20% of the intact protein, retains an altered exonuclease but lacks the polymerase activity of the wild-type enzyme (25). Analysis of the crystal structure of the Klenow fragment of E. coli polymerase I and the enzymatic activities of its mutants indicate that Asp-424 is involved in coordination of a metal ion and is essential for exonucleolytic cleavage (26-29). Amino acid sequence comparisons (6) have suggested that Asp-219 in T4 DNA polymerase is in an analogous position. In this report, we describe the construction and initial characterization of a T4 DNA polymerase mutant with alanine replacing Asp-219. We show that this polymerase mutant has no measurable exonuclease, but retains a polymerase activity whose kinetic parameters are very close to those of the wild type. Bacteriophage T4 with the mutant polymerase gene have a markedly increased mutation frequency.MATERIALS AND METHODS

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.The flap endonuclease-1 (FEN-1) 3 nuclease family is conserved in sequence and structure from bacteriophage to humans. These nucleases play essential roles in DNA replication by removing the RNA primers from lagging strand fragments. In addition, FEN-1-related nucleases are important in long-patch base excision repair and in maintenance of genomic stability. Homozygous knockouts of FEN-1 in mice are lethal to embryos (for review, see Refs. 1-3).Like other family members, bacteriophage T4-encoded RNase H shows 5Ј to 3Ј exonuclease activity on either RNA/ DNA or DNA/DNA duplexes and endonuclease activity on either flap or fork DNA structures (4, 5). T4 rnh deletion mutants give no phage production and accumulate unligated, lagging strand fragments in Escherichia coli hosts with defective polymerase I 5Ј nuclease (6). In addition, the mutants are hypersensitive to UV irradiation and anti-tumor agents (7).FEN-1 family nuclease activities are modulated by interactions with DNA replication clamps (e.g. eukaryotic proliferating cell nuclear antigen and T4 gene 45 clamp) and single-stranded DNA-binding proteins (e.g. eukaryotic replication protein A and T4 gene 32 protein) (5, 8 -12). Human FEN-1 nuclease is also stimulated by interactions with the Werner (13, 14) and Bloom (15) syndrome helicases as well as the Rad9-Rad1-Hus1 (9-1-1) checkpoint clamp (16).T4 RNase H 5Ј nuclease removes a short oligonucleotide (1-4 bases) each time it binds its substrate. T4 32 protein, binding on single-stranded DNA behind the nuclease, increases its processivity so that about 10 -50 short oligonucleotides are removed in a single binding event (5). On nicked sub...