The 5′-Exonuclease Activity of Bacteriophage T4 RNase H Is Stimulated by the T4 Gene 32 Single-stranded DNA-binding Protein, but Its Flap Endonuclease Is Inhibited

Abstract

Bacteriophage T4 RNase H is a 5-to 3-nuclease that has exonuclease activity on RNA⅐DNA and DNA⅐DNA duplexes and can remove the pentamer RNA primers made by the T4 primase-helicase (Hollingsworth, H. C., and Nossal, N. G. (1991) J. Biol. Chem. 266, 1888 -1897; Hobbs, L. J., and Nossal, N. G. (1996) J. Bacteriol. 178, 6772-6777). Here we show that this exonuclease degrades duplex DNA nonprocessively, releasing a single oligonucleotide (nucleotides 1-4) with each interaction with the substrate. Degradation continues nonprocessively until the enzyme stops 8 -11 nucleotides from the 3-end of the substrate. T4 gene 32 single-stranded DNA-binding protein strongly stimulates the exonuclease activity of T4 RNase H, converting it into a processive nuclease that removes multiple short oligonucleotides with a combined length of 10 -50 nucleotides each time it binds to the duplex substrate. 32 protein must bind on singlestranded DNA behind T4 RNase H for processive degradation. T4 RNase H also has a flap endonuclease activity that cuts preferentially on either side of the junction between single-and double-stranded DNA in flap and fork DNA structures. In contrast to the exonuclease, the endonuclease is inhibited completely by 32 protein binding to the single strand of the flap substrate. These results suggest an important role for T4 32 protein in controlling T4 RNase H degradation of RNA primers and adjacent DNA during each lagging strand cycle.During the replication of duplex DNA, the synthesis of the discontinuous lagging strand fragments is initiated by short RNA primers that must subsequently be removed and replaced by DNA. In the bacteriophage T4 system, the pentamer RNA primers made by the T4 primase-helicase are removed by a phage-encoded enzyme with RNase H activity that has been called T4 RNase H (1). Our initial characterization of this nuclease showed that it also had 5Ј-to 3Ј-exonuclease activity on double-stranded (ds) 1 DNA, raising the possibility that some DNA adjacent to the RNA primers is removed from the lagging strand fragments. In this paper we show that interaction between T4 gene 32 protein and RNase H controls how much DNA is removed each time T4 RNase H binds to its substrate.T4 DNA replication is carried out by a multienzyme system in which T4 DNA polymerase (gene 43) is tethered to the template by the gene 45 clamp protein that has been loaded on the DNA by the gene 44/62 protein complex. The gene 32 single-stranded (ss) DNA-binding protein covers unwound DNA at the fork and increases the processivity of the lagging strand polymerase. The primase-helicase composed of the gene 61 primase and gene 41 helicase makes the RNA primers on the lagging strand and unwinds the duplex DNA ahead of the leading strand polymerase. The gene 59 helicase assembly protein facilitates the loading of the primase-helicase, especially on DNA covered with 32 protein. After removal of the primer and perhaps adjacent DNA by RNase H, lagging strand fragments are joined by DNA ligase (gene 30) (for review, see Ref. 2).The ge...

Identification of Residues of T4 RNase H Required for Catalysis and DNA Binding

“…The inactive mutant proteins of T4 RNase H are currently being used for co-crystallization with DNA to define the DNA binding site and the actual role of Mg 2ϩ -1 and the aspartates coordinated to it. 3 Our finding that T4 RNase H proteins with mutations in the residues coordinated through water to Mg 2ϩ -2 either retain their activity (D200N and Y86F) or have reduced activity (D157N) (Figs. 3 and 4) suggests either that Mg 2ϩ -2 can remain bound in the absence of any one of these residues or that this metal ion is not required for catalysis.…”

“…Although simple duplex and flap substrates are hydrolyzed at similar rates by the T4 enzyme, only the flap and fork substrates bind tightly enough to be retarded in mobility shift assays (3). We mutagenized the two most conserved basic residues in and adjoining the disordered region (Lys 87 and Arg 90 ), with the expectation that these changes might have more effect on the binding and degradation of flap than duplex substrates.…”

“…Unless otherwise indicated, the materials and methods are those described in the accompanying paper (3).…”

“…Our recent results indicate that this nuclease performs only one round of processive degradation during each lagging strand cycle, removing 10 -50 nucleotides before polymerase elongates the next Okazaki fragment creating a nick sealed by ligase. 1 T4 RNase H also has a flap endonuclease activity, which cuts preferentially near the junction of the single-and doublestranded DNA of the flap or fork structures removing the 5Ј-single-stranded tail (3). This endonuclease activity of T4 RNase H is inhibited by the gene 32 protein.…”

“…The rates of the 5Ј-to 3Ј-nuclease and flap endonuclease reactions are similar. However, in the absence of Mg 2ϩ ions, T4 RNase H binds 100 times better to flap and fork structures than to double-stranded or nicked DNA (3).…”