Bacteriophage T4 gene 41 helicase and gene 59 helicase-loading protein: a versatile couple with roles in replication and recombination.

Bacteriophage T4 uses two modes of replication initiation: origin-dependent replication early in infection and recombination-dependent replication at later times. The same relatively simple complex of T4 replication proteins is responsible for both modes of DNA synthesis. Thus the mechanism for loading the T4 41 helicase must be versatile enough to allow it to be loaded on R loops created by transcription at several origins, on D loops created by recombination, and on stalled replication forks. T4 59 helicase-loading protein is a small, basic, almost completely alpha-helical protein whose N-terminal domain has structural similarity to high mobility group family proteins. In this paper we review recent evidence that 59 protein recognizes specific structures rather than specific sequences. It binds and loads the helicase on replication forks and on three- and four-stranded (Holliday junction) recombination structures, without sequence specificity. We summarize our experiments showing that purified T4 enzymes catalyze complete unidirectional replication of a plasmid containing the T4 ori(uvsY) origin, with a preformed R loop at the position of the R loop identified at this origin in vivo. This replication depends on the 41 helicase and is strongly stimulated by 59 protein. Moreover, the helicase-loading protein helps to coordinate leading and lagging strand synthesis by blocking replication on the ori(uvsY) R loop plasmid until the helicase is loaded. The T4 enzymes also can replicate plasmids with R loops that do not have a T4 origin sequence, but only if the R loops are within an easily unwound DNA sequence.

Bacteriophage T4 RNase H removes both RNA primers and adjacent DNA from the 5' end of lagging strand fragments.

Bacteriophage T4 RNase H belongs to a family of prokaryotic and eukaryotic nucleases that remove RNA primers from lagging strand fragments during DNA replication. Each enzyme has a flap endonuclease activity, cutting at or near the junction between single- and double-stranded DNA, and a 5'- to 3'-exonuclease, degrading both RNA.DNA and DNA.DNA duplexes. On model substrates for lagging strand synthesis, T4 RNase H functions as an exonuclease removing short oligonucleotides, rather than as an endonuclease removing longer flaps created by the advancing polymerase. The combined length of the DNA oligonucleotides released from each fragment ranges from 3 to 30 nucleotides, which corresponds to one round of processive degradation by T4 RNase H with 32 single-stranded DNA-binding protein present. Approximately 30 nucleotides are removed from each fragment during coupled leading and lagging strand synthesis with the complete T4 replication system. We conclude that the presence of 32 protein on the single-stranded DNA between lagging strand fragments guarantees that the nuclease will degrade processively, removing adjacent DNA as well as the RNA primers, and that the difference in the relative rates of synthesis and hydrolysis ensures that there is usually only a single round of degradation during each lagging strand cycle.

Bacteriophage T4 proteins replicate plasmids with a preformed R loop at the T4 ori(uvsY) replication origin in vitro.

Bacteriophage T4 DNA replication proteins catalyze complete unidirectional replication of plasmids containing the T4 ori(uvsY) replication origin in vitro, beginning with a preformed R loop at the position of the origin R loop previously identified in vivo. T4 DNA polymerase, clamp, clamp loader, and 32 protein are needed for initial elongation of the RNA, which serves as the leading-strand primer. Normal replication is dependent on T4 41 helicase and 61 primase and is strongly stimulated by the 59 helicase loading protein. 59 protein slows replication without the helicase. As expected, leading-strand synthesis stalls prematurely in the absence of T4 DNA topoisomerase. A DNA unwinding element (DUE) is essential for replication, but the ori(uvsY) DUE can be replaced by other DUE sequences.

Analysis of the Okazaki fragment distributions along single long DNAs replicated by the bacteriophage T4 proteins.

Rolling circle replication from M13 DNA circles was previously reconstituted in vitro using purified factors encoded by bacteriophage T4. The products are duplex circles with linear tails >100 kb. When T4 DNA polymerase deficient in 3' to 5' exonuclease activity was employed, electron microscopy revealed short single-stranded DNA "flaps" along the replicated tails. This marked the beginning of each Okazaki fragment, allowing an analysis of the lengths of sequential Okazaki fragments on individual replicating molecules. DNAs containing runs of Okazaki fragments of similar length were found, but most showed large length variations over runs of six or more fragments reflecting the broad population distribution.

Interaction of the bacteriophage T4 gene 59 helicase loading protein and gene 41 helicase with each other and with fork, flap, and cruciform DNA.

Bacteriophage T4 gene 59 helicase loading protein accelerates the loading of T4 gene 41 DNA helicase and is required for recombination-dependent DNA replication late in T4 phage infection. The crystal structure of 59 protein revealed a two-domain alpha-helical protein, whose N-terminal domain has strong structural similarity to the DNA binding domain of high mobility group family proteins (Mueser, T. C., Jones, C. E., Nossal, N. G., and Hyde, C. C. (2000) J. Mol. Biol. 296, 597-612). We have previously shown that 59 protein binds preferentially to fork DNA. Here we show that 59 protein binds to completely duplex forks but cannot load the helicase unless there is a single-stranded gap of more than 5 nucleotides on the fork arm corresponding to the lagging strand template. Consistent with the roles of these proteins in recombination, we find that 59 protein binds to and stimulates 41 helicase activity on Holliday junction DNA, and on a substrate that resembles a strand invasion structure. 59 protein forms a stable complex with wild type 41 helicase and fork DNA in the presence of adenosine 5'-O-(thiotriphosphate). The unwinding activity of 41 helicase missing 20 C-terminal amino acids is not stimulated by 59 protein, and it does not form a complex with 59 protein on fork DNA.

Bacteriophage T4 gene 59 helicase assembly protein binds replication fork DNA. The 1.45 A resolution crystal structure reveals a novel alpha-helical two-domain fold.

The bacteriophage T4 gene 59 helicase assembly protein is required for recombination-dependent DNA replication, which is the predominant mode of DNA replication in the late stage of T4 infection. T4 gene 59 helicase assembly protein accelerates the loading of the T4 gene 41 helicase during DNA synthesis by the T4 replication system in vitro. T4 gene 59 helicase assembly protein binds to both T4 gene 41 helicase and T4 gene 32 single-stranded DNA binding protein, and to single and double-stranded DNA. We show here that T4 gene 59 helicase assembly protein binds most tightly to fork DNA substrates, with either single or almost entirely double-stranded arms. Our studies suggest that the helicase assembly protein is responsible for loading T4 gene 41 helicase specifically at replication forks, and that its binding sites for each arm must hold more than six, but not more than 12 nucleotides. The 1.45 A resolution crystal structure of the full-length 217-residue monomeric T4 gene 59 helicase assembly protein reveals a novel alpha-helical bundle fold with two domains of similar size. Surface residues are predominantly basic (pI 9.37) with clusters of acidic residues but exposed hydrophobic residues suggest sites for potential contact with DNA and with other protein molecules. The N-terminal domain has structural similarity to the double-stranded DNA binding domain of rat HMG1A. We propose a speculative model of how the T4 gene 59 helicase assembly protein might bind to fork DNA based on the similarity to HMG1, the location of the basic and hydrophobic regions, and the site size of the fork arms needed for tight fork DNA binding. The fork-binding model suggests putative binding sites for the T4 gene 32 single-stranded DNA binding protein and for the hexameric T4 gene 41 helicase assembly.

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.

Bacteriophage T4 RNase H is a 5'- to 3'-nuclease that has exonuclease activity on RNA.DNA and DNA.DNA 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 single-stranded 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.

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.

Role of the bacteriophage T7 and T4 single-stranded DNA-binding proteins in the formation of joint molecules and DNA helicase-catalyzed polar branch migration.

Bacteriophage T7 gene 2.5 single-stranded DNA-binding protein and gene 4 DNA helicase together promote pairing of two homologous DNA molecules and subsequent polar branch migration (Kong, D., and Richardson, C. C. (1996) EMBO J. 15, 2010-2019). In this report, we show that gene 2.5 protein is not required for the initiation or propagation of strand transfer once a joint molecule has been formed between the two DNA partners, a reaction that is mediated by the gene 2.5 protein alone. A mutant gene 2.5 protein, gene 2.5-Delta21C protein, lacking 21 amino acid residues at its C terminus, cannot physically interact with gene 4 protein. Although it does bind to single-stranded DNA and promote the formation of joint molecule via homologous base pairing, subsequent strand transfer by gene 4 helicase is inhibited by the presence of the gene 2.5-Delta21C protein. Bacteriophage T4 gene 32 protein likewise inhibits T7 gene 4 protein-mediated strand transfer, whereas Escherichia coli single-stranded DNA-binding protein does not. The 63-kDa gene 4 protein of phage T7 is also a DNA primase in that it catalyzes the synthesis of oligonucleotides at specific sequences during translocation on single-stranded DNA. We find that neither the rate nor extent of strand transfer is significantly affected by concurrent primer synthesis. The bacteriophage T4 gene 41 helicase has been shown to catalyze polar branch migration after the T4 gene 59 helicase assembly protein loads the helicase onto joint molecules formed by the T4 UvsX and gene 32 proteins (Salinas, F., and Kodadek, T. (1995) Cell 82, 111-119). We find that gene 32 protein alone forms joint molecules between partially single-stranded homologous DNA partners and that subsequent branch migration requires this single-stranded DNA-binding protein in addition to the gene 41 helicase and the gene 59 helicase assembly protein. Similar to the strand transfer reaction, strand displacement DNA synthesis catalyzed by T4 DNA polymerase also requires the presence of gene 32 protein in addition to the gene 41 and 59 proteins.

Either bacteriophage T4 RNase H or Escherichia coli DNA polymerase I is essential for phage replication.

Bacteriophage T4 rnh encodes an RNase H that removes ribopentamer primers from nascent DNA chains during synthesis by the T4 multienzyme replication system in vitro (H. C. Hollingsworth and N. G. Nossal, J. Biol. Chem. 266:1888-1897, 1991). This paper demonstrates that either T4 RNase HI or Escherichia coli DNA polymerase I (Pol I) is essential for phage replication. Wild-type T4 phage production was not diminished by the polA12 mutation, which disrupts coordination between the polymerase and the 5'-to-3' nuclease activities of E. coli DNA Pol I, or by an interruption in the gene for E. coli RNase HI. Deleting the C-terminal amino acids 118 to 305 from T4 RNase H reduced phage production to 47% of that of wild-type T4 on a wild-type E. coli host, 10% on an isogenic host defective in RNase H, and less than 0.1% on a polA12 host. The T4 rnh(delta118-305) mutant synthesized DNA at about half the rate of wild-type T4 in the polA12 host. More than 50% of pulse-labelled mutant DNA was in short chains characteristic of Okazaki fragments. Phage production was restored in the nonpermissive host by providing the T4 rnh gene on a plasmid. Thus, T4 RNase H was sufficient to sustain the high rate of T4 DNA synthesis, but E. coli RNase HI and the 5'-to-3' exonuclease of Pol I could substitute to some extent for the T4 enzyme. However, replication was less accurate in the absence of the T4 RNase H, as judged by the increased frequency of acriflavine-resistant mutations after infection of a wild-type host with the T4 rnh (delta118-305) mutant.

Structure of bacteriophage T4 RNase H, a 5' to 3' RNA-DNA and DNA-DNA exonuclease with sequence similarity to the RAD2 family of eukaryotic proteins.

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.

A single mutation in bacteriophage T4 DNA polymerase (A737V, tsL141) decreases its processivity as a polymerase and increases its processivity as a 3'-->5' exonuclease.

The bacteriophage T4 DNA polymerase mutant A737V (tsL141 and tsCB120) was originally characterized as temperature-sensitive for DNA replication and an antimutator for transition mutations. Its antimutator phenotype is suppressed by the L771F mutation (Reha-Krantz, L. J., Stocki, S., Nonay, R., and Maughan, C. (1989) J. Cell. Biochem. 13D, 140). We find that the A737V polymerase arrests much more frequently than the wild type when polymerizing on primed single-stranded DNA templates. Although the 3'-->5' exonuclease of the mutant is indistinguishable from the wild type on single-stranded DNA, it is more active than the wild type on duplex DNA. In a single encounter with the primer, the wild type polymerase can incorporate more than 50 nucleotides. The processivity of the A737V polymerase is less than the wild type as a polymerase, but is greater than the wild type as an exonuclease. The L771F polymerase resembles the wild type in each of these properties, while the double mutant (A737V, L771F) is intermediate between the two single mutants. Kinetic studies of wild type T4 DNA polymerase (Capson, T. L., Peliska, J. A., Kaboord, B. F., Frey, M. W., Lively, C., Dahlberg, M., and Benkovic, S. J. (1992) Biochemistry 31, 10984-10994) suggest that DNA binds first to the polumerase active site, before adopting a configuration in which it can be hydrolyzed by the exonuclease. Within this framework, our studies suggest that DNA moves more readily from the polymerase- to the exonuclease-competent configuration on the A737V mutant polymerase, and that this movement is decreased by the compensating L771F mutation.

Interaction of DNA polymerase and DNA helicase within the bacteriophage T4 DNA replication complex. Leading strand synthesis by the T4 DNA polymerase mutant A737V (tsL141) requires the T4 gene 59 helicase assembly protein.

The bacteriophage T4 tsL141 (A737V) mutant in T4 DNA polymerase is temperature-sensitive for DNA replication and an antimutator for some types of mutations. In the accompanying paper (Spacciapoli, P., and Nossal, N. G. (1993) J. Biol. Chem. 269, 438-446), we show that the purified A737V T4 DNA polymerase is less processive than the wild type enzyme as a polymerase, but is more processive as an exonuclease. The bacteriophage T4 multienzyme replication complex reconstituted with the A737V mutant polymerase is defective in both lagging and leading strand synthesis. On lagging strand templates, the A737V polymerase is stimulated by the gene 44/62 and 45 polymerase accessory proteins and the gene 32 DNA binding protein, but is still arrested at pause sites much more frequently than the wild type. In contrast to wild type T4 DNA polymerase, the A737V polymerase does not catalyze leading strand synthesis on a forked duplex template with the polymerase accessory proteins, 32 protein, and the gene 41 protein helicase. The A737V polymerase requires the T4 gene 59 helicase assembly protein, as well as the other proteins, to carry out this reaction. Each of these defects is suppressed by the intragenic L771F mutation that suppresses the antimutator phenotype of the A737V, polymerase in vivo (Reha-Krantz, L. J., Stocki, S., Nonay, R., and Maughan, C. (1989) J. Cell. Biochem. 13D, 140).

Construction and characterization of a bacteriophage T4 DNA polymerase deficient in 3'-->5' exonuclease activity.

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 10(7), but it retains a polymerase activity whose kinetic parameters, kcat, Kd DNA, and Kd dATP, are very close to those of the wild-type enzyme. Bacteriophage T4 with the mutant polymerase gene has a markedly increased mutation frequency. Asp-219 in T4 DNA polymerase is within a sequence similar to those surrounding Asp residues previously shown to be essential for the exonuclease activities of the Klenow fragment of Escherichia coli DNA polymerase I (Asp-424), bacteriophage phi 29 DNA polymerase (Asp-66), and Saccharomyces cerevisiae DNA polymerase delta (Asp-405). Thus, these studies support the proposal that there are similar sequences in the active sites for the proofreading exonucleases of these and related DNA polymerases.

Protein-protein interactions at a DNA replication fork: bacteriophage T4 as a model.

The DNA replication system of bacteriophage T4 serves as a relatively simple model for the types of reactions and protein-protein interactions needed to carry out and coordinate the synthesis of the leading and lagging strands of a DNA replication fork. At least 10 phage-encoded proteins are required for this synthesis: T4 DNA polymerase, the genes 44/62 and 45 polymerase accessory proteins, gene 32 single-stranded DNA binding protein, the genes 61, 41, and 59 primase-helicase, RNase H, and DNA ligase. Assembly of the polymerase and the accessory proteins on the primed template is a stepwise process that requires ATP hydrolysis and is strongly stimulated by 32 protein. The 41 protein helicase is essential to unwind the duplex ahead of polymerase on the leading strand, and to interact with the 61 protein to synthesize the RNA primers that initiate each discontinuous fragment on the lagging strand. An interaction between the 44/62 and 45 polymerase accessory proteins and the primase-helicase is required for primer synthesis on 32 protein-covered DNA. Thus it is possible that the signal for the initiation of a new fragment by the primase-helicase is the release of the polymerase accessory proteins from the completed adjacent fragment.

Protein-DNA cross-linking demonstrates stepwise ATP-dependent assembly of T4 DNA polymerase and its accessory proteins on the primer-template.

T4 DNA polymerase, the 44/62 and 45 polymerase accessory proteins, and 32 single-stranded DNA-binding protein catalyze ATP-dependent DNA synthesis. Using DNA primers with cross-linkable residues at specific positions, we obtained structural data that reveal how these proteins assemble on the primer-template. With the nonhydrolyzable ATP analog ATP gamma S, assembly of the 44/62 and 45 proteins on the primer requires 32 protein but not polymerase. ATP hydrolysis changes the position and intensity of cross-linking to each of the accessory proteins and allows cross-linking of polymerase. Our data indicate that the initial binding of the three accessory proteins and ATP to a 32 protein-covered primer-template is followed by ATP hydrolysis, binding of polymerase, and movement of the accessory proteins to yield a complex capable of processive DNA synthesis.

Bacteriophage T4 encodes an RNase H which removes RNA primers made by the T4 DNA replication system in vitro.

RNase H activity increases markedly after bacteriophage T4 infection of Escherichia coli MIC2003, an RNase H-deficient host. We have extensively purified the RNase H from these T4-infected cells and have shown that the RNase H activity copurifies with a 5' to 3' DNA exonuclease activity. The N-terminal sequence of a 35-kDa protein copurifying with the RNase H activity matches the terminus of the predicted product of an open reading frame (designated ORF A or 33.2) upstream of T4 gene 33, identified previously by Hahn and co-workers (Hahn, S., Kruse, U., and Rüger, W. (1986) Nucleic Acids Res. 14, 9311-9327). Plasmids containing ORF A under the control of the T7 promoter express RNase H and 5' to 3' DNA exonuclease activities as well as a protein that comigrates on sodium dodecyl sulfate-polyacrylamide gels with the 35-kDa protein present in the RNase H purified from T4-infected cells. T4 RNase H removes the pentamer RNA primers from DNA chains initiated by the T4 primase-helicase (gene products 61 and 41). Addition of T4 RNase H and T4 DNA ligase leads to extensive joining of discontinuous lagging strand fragments in the T4 DNA replication system in vitro.