Template recognition sequence for RNA primer synthesis by gene 4 protein of bacteriophage T7.

Replication of DNA plays a central role in transmitting hereditary information from cell to cell. To achieve reliable DNA replication, multiple proteins form a stable complex, known as the replisome, enabling them to act together in a highly coordinated fashion. Over the past decade, the roles of the various proteins within the replisome have been determined. Although many of their interactions have been characterized, it remains poorly understood how replication proteins enter and leave the replisome. In this study, we visualize fluorescently labeled bacteriophage T7 DNA polymerases within the replisome while we simultaneously observe the kinetics of the replication process. This combination of observables allows us to monitor both the activity and dynamics of individual polymerases during coordinated leading-and lagging-strand synthesis. Our data suggest that lagging-strand polymerases are exchanged at a frequency similar to that of Okazaki fragment synthesis and that two or more polymerases are present in the replisome during DNA replication. Our studies imply a highly dynamic picture of the replisome with lagging-strand DNA polymerases residing at the fork for the synthesis of only a few Okazaki fragments. Further, new lagging-strand polymerases are readily recruited from a pool of polymerases that are proximally bound to the replisome and continuously replenished from solution.polymerase exchange | single molecule | fluorescence microscopy T he organization of replisomes is highly conserved among various organisms (1), underlining the evolutionary importance of the replication machinery architecture. The bacteriophage T7 replication system offers an attractive model system to study the interplay between replication proteins because its replication machinery is relatively simple; a functional replisome can be reconstituted by just four purified proteins. Three of these proteins are encoded by the phage itself: helicase-primase (gp4), DNA polymerase (gp5), and single-stranded DNA (ssDNA) binding protein (gp2.5). A processivity factor for the gp5 polymerase, thioredoxin (trx), is provided by the host Escherichia coli.

Section: Resultsmentioning

confidence: 99%

Single-molecule studies of polymerase dynamics and stoichiometry at the bacteriophage T7 replication machinery

Geertsema

Kulczyk

Richardson

et al. 2014

“…Products are indicated to the right of the gel image. Pentamers are likely not extended efficiently (37,38). (C ) Klenow fragment of E. coli DNA polymerase I and T4 DNA polymerase cannot extend short RNAs synthesized by gp4.…”

Section: Significancementioning

confidence: 99%

Primer release is the rate-limiting event in lagging-strand synthesis mediated by the T7 replisome

Hernandez

Lee

Richardson

2016

DNA replication occurs semidiscontinuously due to the antiparallel DNA strands and polarity of enzymatic DNA synthesis. Although the leading strand is synthesized continuously, the lagging strand is synthesized in small segments designated Okazaki fragments. Lagging-strand synthesis is a complex event requiring repeated cycles of RNA primer synthesis, transfer to the lagging-strand polymerase, and extension effected by cooperation between DNA primase and the lagging-strand polymerase. We examined events controlling Okazaki fragment initiation using the bacteriophage T7 replication system. Primer utilization by T7 DNA polymerase is slower than primer formation. Slow primer release from DNA primase allows the polymerase to engage the complex and is followed by a slow primer handoff step. The T7 single-stranded DNA binding protein increases primer formation and extension efficiency but promotes limited rounds of primer extension. We present a model describing Okazaki fragment initiation, the regulation of fragment length, and their implications for coordinated leading-and lagging-strand DNA synthesis.Okazaki fragment | DNA primase | replisome | primer

“…4 B and C). In these experiments we examined oligonucleotide synthesis on M13 DNA where all three major primase recognition sites are present as well as minor recognition sites (19). As with wild-type gp4 the altered primase synthesizes tetraribonucleotides as well as the precursors, di-and trinucleotides (Fig.…”

Section: Substitution Of Lysine For Tryptophan At Position 69 Selectimentioning

confidence: 99%

“…and 5′-d(GTGTC)-3′ to catalyze the synthesis of functional tetraribonucleotides, r(ACCC), r(ACCA), and r(ACAC) that the lagging strand DNA polymerase can use to initiate the synthesis of Okazaki fragments (19,20). The 56-kDa gp4 cannot catalyze template-dependent tetraribonucleotide synthesis because it lacks the ZBD.…”

mentioning

confidence: 99%

Direct role for the RNA polymerase domain of T7 primase in primer delivery

Zhu

Lee²,

Richardson³

2010

Gene 4 protein (gp4) encoded by bacteriophage T7 contains a C-terminal helicase and an N-terminal primase domain. After synthesis of tetraribonucleotides, gp4 must transfer them to the polymerase for use as primers to initiate DNA synthesis. In vivo gp4 exists in two molecular weight forms, a 56-kDa form and the full-length 63-kDa form. The 56-kDa gp4 lacks the N-terminal Cys 4 zinc-binding motif important in the recognition of primase sites in DNA. The 56-kDa gp4 is defective in primer synthesis but delivers a wider range of primers to initiate DNA synthesis compared to the 63-kDa gp4. Suppressors exist that enable the 56-kDa gp4 to support the growth of T7 phage lacking gene 4 (T7Δ4). We have identified 56-kDa DNA primases defective in primer delivery by screening for their ability to support growth of T7Δ4 phage in the presence of this suppressor. Trp69 is critical for primer delivery. Replacement of Trp69 with lysine in either the 56-or 63-kDa gp4 results in defective primer delivery with other functions unaffected. DNA primase harboring lysine at position 69 fails to stabilize the primer on DNA. Thus, a primase subdomain not directly involved in primer synthesis is involved in primer delivery. The stabilization of the primer by DNA primase is necessary for DNA polymerase to initiate synthesis.T7 bacteriophage | primer handoff D NA polymerases cannot initiate synthesis of DNA de novo, requiring a 3′-hydroxyl terminated primer positioned at the catalytic site. During DNA replication, primers are synthesized by a class of enzymes designated DNA primases (1). DNA primases catalyze the synthesis of short oligoribonucleotides that can then be used as primers by DNA polymerase. DNA primases play roles in the loading of DNA helicase (2), temporal regulation of replication (3-5), and handoff of the primer to the DNA polymerase (6-8).Based on their sequences and structures, DNA primases can be grouped into two families-the prokaryotic and the eukaryotic/ archaeal primases (1). Prokaryotic primases (see Fig. 1A for T7 DNA primase) contain six conserved sequence motifs (9). An N-terminal zinc-binding domain (ZBD) formed by motif I is a determinant for recognition of a specific sequence in DNA (10-13). Motifs II-VI are located in the C-terminal RNA polymerase domain (RPD) that consists of two subdomains (Fig. 1A). The C-terminal topoisomerase-primase (TOPRIM) fold (14) contains the active site where the metal-mediated condensation of nucleotides occurs. The N-terminal subdomain of the RPD is less conserved (6), and its role remains elusive. A recent study suggests that this subdomain contains a ssDNA binding groove through which the template can track (15).Gene 4 of bacteriophage T7 encodes two colinear proteins. The full-length product is a 63-kDa protein (63-kDa gp4) in which the C-terminal half encodes the helicase and the N-terminal half the primase (Fig. 1B). The coexistence of both primase and helicase in a single polypeptide is unique in that the two activities are physically associated in other systems but are e...