For a single replisome to mediate these multiple reac-* Department of Biological Chemistry and tions, it is necessary to loop the lagging strand back Molecular Pharmacology through the replisome; otherwise, reactions occurring Harvard Medical School at the 3Ј terminus of the growing Okazaki fragment Boston, Massachusetts 02115 would be hundreds of nucleotides distal to leading † Lineberger Comprehensive Cancer Center strand events. Such a "trombone" model was first pro-University of North Carolina at Chapel Hill posed by Alberts et al. for the bacteriophage T4 system Chapel Hill, North Carolina 27599-7295 (Alberts et al., 1975) and was subsequently extended to the Escherichia coli (Arai and Kornberg, 1981;Wu et al., 1994) and phage T7 systems (Debyser et al., 1994). In all of these models, a dimeric DNA polymerase extends Summary both strands in the same overall direction and at the same rate. Kinetic data and the size of Okazaki frag-The coordinated synthesis of both leading and lagging ments have provided indirect evidence for coordination DNA strands is thought to involve a dimeric DNA polyof leading and lagging strand synthesis in both the E. merase and a looping of the lagging strand so that coli (Wu et al., 1994; Kim et al., 1996; Tougu and Marians, both strands can be synthesized in the same direction.1996) and phage systems (Alberts et al., 1983; Debyser We have constructed a minicircle with a replication et al., 1994). More direct evidence has come from the fork that permits an assessment of the stoichiometry dimeric structure of E. coli DNA polymerase III (Maki et of the proteins and a measurement of the synthesis McHenry, 1991;Onrust et al., 1995; Yuzhakov of each strand. The replisome consisting of bacterio et al., 1996) and a demonstration that both polymerases phage T7 DNA polymerase, helicase, primase, and sincan function simultaneously within the holoenzyme gle-stranded DNA-binding protein mediates coordicomplex (Yuzhakov et al., 1996). Recently, using a prenated replication. The criteria for coordination are formed replication fork on a duplex M13 DNA molecule, fulfilled: (1) a replication loop is formed, (2) leading and loops of lagging strand DNA were observed by electron lagging strand synthesis are coupled, (3) the lagging microscopy in the T7 replication system (Park et al., strand polymerase recycles from one Okazaki frag-1998), providing evidence for the postulated replication ment to another, and (4) the length of Okazaki fragloop. ments is regulated. T7 single-stranded DNA-bindingIn spite of this body of evidence, an unambiguous protein is essential for coordination.demonstration of a functioning replisome has been an elusive goal. In the present study, we have used the

The gene 4 proteins of bacteriophage T7 provide both primase and helicase activities at the replication fork. Efficient DNA replication requires that the functions of the gene 4 protein be coordinated with the movement of the T7 DNA polymerase. We show that a carboxyl-terminal domain of the gene 4 protein is required for interaction with T7 DNA polymerase during leading strand DNA synthesis. The carboxyl terminus of the gene 4 protein is highly acidic: of the 17 carboxyl-terminal amino acids 7 are negatively charged. Deletion of the coding region for these 17 residues results in a gene 4 protein that cannot support the growth of T7 phage. The purified mutant gene 4 protein has wild-type levels of both helicase and primase activities; however, DNA synthesis catalyzed by T7 DNA polymerase on a duplex DNA substrate is stimulated by this mutant protein to only about 5% of the level of synthesis obtained with wildtype protein. The mutant gene 4 protein can form hexamers and bind single-stranded DNA, but as determined by native PAGE analysis, the protein cannot form a stable complex with the DNA polymerase. The mutant gene 4 protein can prime DNA synthesis normally, indicating that for lagging strand synthesis a different set of helicase/primase-DNA polymerase interactions are involved. These findings have implications for the mechanisms coupling leading and lagging strand DNA synthesis at the T7 replication fork.

No abstract

No abstract

Absolute homology between partner substrates within the strand exchange region is an essential requirement for recombination mediated by the yeast site-specific recombinase Flp. Using combinations of specially designed half- and full-site Flp substrates, we demonstrate that the strand joining step of recombination is exquisitely sensitive to spacer homology. At each exchange point, 2-3 spacer nucleotides adjacent to the nick within the cleaved strand of one substrate must base pair with the corresponding segment of the un-nicked strand from the second substrate for efficient strand joining in the recombinant mode. In accordance with the "cis-activation/trans-nucleophilic attack" model for each of the two transesterification steps of Flp recombination (strand cleavage and strand joining), we propose that the limited strand pairing orients the DNA-nucleophile (5'-hydroxyl) for attack on its target diester (3'-phosphotyrosyl-Flp). During one round of recombination, 4-6 terminal base pairs of the spacer (2-3 base pairs at each spacer end) must unpair, following strand cleavage, within a DNA substrate and pair with the partner substrate prior to strand union. In this model, the extent of branch migration of the covalently closed Holliday intermediate is limited to the central core of the spacer. The templated positioning of reactive nucleic acid groups (which is central to the model) may be utilized by other recombination systems and by RNA splicing reactions.