Coordinated Leading and Lagging Strand DNA Synthesis on a Minicircular Template

Abstract

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 Linker Region between the Helicase and Primase Domains of the Gene 4 Protein of Bacteriophage T7

“…The complete reaction (20 l) contained 1 M gene 4 protein, 1 mM ␤,␥-methylene dTTP, and 0.1 M 45-mer oligonucleotide (5Ј-AGAGC GTCAC TCTTG TGACT ACCAG TGGTC GCAAA GTTCT TATCT-3Ј). After incubation for 20 min at 37°C, the reaction mixture was loaded onto a 10% non-denaturing polyacrylamide gel and electrophoresed at 4°C for 5 h. Gel running buffer (25 mM Tris-HCl, pH 7.0, 190 mM glycine) contained 10 mM Mg(OAc) 2 or did not. The protein was stained with Coomassie Blue in order to visualize the oligomerized protein.…”

“…The replisome of bacteriophage T7 is unique in that the proteins that account for these five functions mediate coordinated leading and lagging strand DNA synthesis without the aid of other accessory proteins such as clamp-loading proteins and helicaseloading proteins (2). All of these activities with the exception of the processivity factor are encoded by the phage.…”

Genesis and Wanderings: Origins and Migrations in Asymmetrically Replicating Mitochondrial DNA

“…In symmetrically replicating DNA, such as in E. coli and bacteriophage, lagging-strand synthesis is thought to occur by looping of the lagging-strand template back to the leading-strand replication fork. 10 These loops are large, and average in the range of 1-3 Kb. 11 Although there are important similarities between T-odd bacteriophage and mitochondrial replication proteins, single-strand base pairing and stem formation is not a characteristic of the bacterial loops.…”

A Molecular Handoff between Bacteriophage T7 DNA Primase and T7 DNA Polymerase Initiates DNA Synthesis

“…Phage T7 replicates DNA using four proteins, T7 gene 4 primase-helicase protein (gp4), T7 gene 2.5 ssDNA-binding protein (gp2.5), and T7 DNA polymerase (gp5) complexed with its processivity factor, Escherichia coli thioredoxin (4). The reactions catalyzed by the individual proteins are coordinated during replication by the assembly of a multiprotein complex that moves with the replication fork (5,6). During replication, the primase-helicase directly contacts both the DNA polymerase and gp2.5 (7)(8)(9)(10).…”

“…A C-terminal acidic segment of the primase-helicase is required for its interaction with the DNA polymerase (10). An interaction between gp2.5 and T7 DNA polymerase is required for the coordinated synthesis of both leading and lagging strands of the replication fork (5,6). A C-terminal 21-residue region of gp2.5 is essential not only for the interactions with the DNA polymerase and the primase-helicase but also for dimerization of gp2.5 (9).…”