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Primer Release Is the Rate-Limiting Event in Lagging-Strand Synthesis

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Primer Release Is the Rate-Limiting Event in Lagging-Strand Synthesis Primer release is the rate-limiting event in lagging- strand synthesis mediated by the T7 replisome Alfredo J. Hernandeza, Seung-Joo Leea, and Charles C. Richardsona,1 aDepartment of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115 Contributed by Charles C. Richardson, April 18, 2016 (sent for review December 30, 2015; reviewed by Nicholas E. Dixon and I. Robert Lehman) DNA replication occurs semidiscontinuously due to the antiparallel but also for enabling the use of short oligoribonucleotides by T7 DNA strands and polarity of enzymatic DNA synthesis. Although DNA polymerase. Critically, the primase domain also fulfills two the leading strand is synthesized continuously, the lagging strand additional roles apart from primer synthesis: it prevents disso- is synthesized in small segments designated Okazaki fragments. ciation of the extremely short tetramer, stabilizing it with the Lagging-strand synthesis is a complex event requiring repeated template, and it secures it in the polymerase active site (10, 12). cycles of RNA primer synthesis, transfer to the lagging-strand Here we show that the rate-limiting step in initiation of Okazaki polymerase, and extension effected by cooperation between DNA fragments by the T7 replisome is primer release from the primase primase and the lagging-strand polymerase. We examined events domain of gp4. In the absence of gp2.5, an additional step, distinct controlling Okazaki fragment initiation using the bacteriophage from primer release, also limits primer extension. The presence of T7 replication system. Primer utilization by T7 DNA polymerase is gp2.5 promotes efficient primer formation and primer utilization. slower than primer formation. Slow primer release from DNA primase allows the polymerase to engage the complex and is Finally, we propose a model for events controlling Okazaki frag- followed by a slow primer handoff step. The T7 single-stranded ment initiation, length, and coordination with synthesis of the DNA binding protein increases primer formation and extension leading strand. efficiency but promotes limited rounds of primer extension. We Results present a model describing Okazaki fragment initiation, the reg- ulation of fragment length, and their implications for coordinated The Use of Short Oligoribonucleotides as Primers by T7 DNA leading- and lagging-strand DNA synthesis. Polymerase Is Dependent on gp4. T7 gp4 synthesizes tetra- ribonucleotides from ATP and CTP in the presence of divalent Okazaki fragment | DNA primase | replisome | primer cations and ssDNA containing a PRS (Fig. 1 A and B). In the presence of T7 DNA polymerase, the tetraribonucleotides eplicative DNA polymerases require a primer for initiation are extended by incorporation of deoxyribonucleotides in a R(1, 2). Although various priming strategies exist, the most template-dependent manner (Fig. 1B). Although the primase ubiquitous involves use of short RNAs synthesized by DNA pri- domain of gp4 is alone sufficient for primer synthesis (13), it mases. Although the leading strand is synthesized continuously in enables their use by T7 DNA polymerase ineffectively. The the direction of replication fork movement, the lagging strand is enhanced use of primers by full-length gp4 suggests that the synthesized in small segments called Okazaki fragments that are polymerase engages gp4 through contacts with the primase and later joined together. Initiation of Okazaki fragment synthesis is a helicase domains or the helicase domain efficiently tethers the complex, tightly regulated process involving multiple enzymatic primase domain to DNA. The requirement for a physical in- events and molecular interactions (1, 3). teraction between gp4 and T7 DNA polymerase to promote The replication machinery of bacteriophage T7 is among the primer extension (14, 15) is underscored by the inability of simplest replication systems (4, 5). Only four proteins are required other polymerases, such as T4 DNA polymerase or the Klenow to reconstitute coordinated DNA synthesis in vitro: gene 4 primase- fragment of E. coli DNA polymerase I, to extend primers helicase (gp4) unwinds the DNA duplex to provide the template synthesized by gp4 (Fig. 1B). for DNA synthesis. T7 DNA polymerase (gp5), in complex with its processivity factor, Escherichia coli thioredoxin (Trx), is responsible Significance for synthesis of leading and lagging strands. Finally, gene 2.5 single- stranded (ss)DNA-binding protein (gp2.5) stabilizes ssDNA repli- cation intermediates and is essential for coordination of DNA Lagging-strand DNA is replicated in multiple segments called synthesis on both strands. The elegant simplicity of the T7 repli- Okazaki fragments, whose formation involves a complex mo- cation machinery makes it an attractive system for investigating lecular cycle mediated by DNA primase, polymerase, and other molecular and enzymatic events occurring during DNA replication. replisome components. In addition, synthesis of the lagging In T7-infected E. coli, Okazaki fragments are initiated by syn- strand must occur in lockstep with the leading strand. Using thesis of tetraribonucleotides by the primase activity of gp4 (6) the simple replication system of bacteriophage T7, we found (Fig. 1A). Gp4 catalyzes the formation of tetraribonucleotides at that primer release from the DNA primase domain of T7 pri- specific template sequences, designated “primase recognition sites” mase helicase is a critical regulatory event in the initiation of (PRSs) (7). On encountering a 5′-GTC-3′ sequence, gp4 catalyzes Okazaki fragments and that the T7 single-stranded binding the synthesis of the dinucleotide pppAC. The “cryptic” cytosine in protein, gp2.5, regulates initiation timing. the recognition site is not copied into the oligoribonucleotide. The Author contributions: A.J.H. and C.C.R. designed research; A.J.H. performed research; dinucleotide is extended to a trinucleotide, and finally, to the A.J.H. and S.-J.L. contributed new reagents/analytic tools; A.J.H. analyzed data; and A.J.H., functional tetraribonucleotide primers, pppACCC, pppACCA, or S.-J.L., and C.C.R. wrote the paper. pppACAC if the appropriate complementary sequence is present Reviewers: N.E.D., University of Wollongong; and I.R.L., Stanford University School (8). Once primers are synthesized, they are delivered to the lagging- of Medicine. strand polymerase (9–11). T7 DNA polymerase alone cannot The authors declare no conflict of interest. efficiently use primers shorter than 15 nt in vitro. However, in 1To whom correspondence should be addressed. Email: [email protected]. the presence of gp4, it uses tetramers as primers for DNA syn- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. thesis. Therefore, gp4 is critical not only for primer formation, 1073/pnas.1604894113/-/DCSupplemental. 5916–5921 | PNAS | May 24, 2016 | vol. 113 | no. 21 www.pnas.org/cgi/doi/10.1073/pnas.1604894113 Downloaded by guest on September 27, 2021 − of ∼0.03 s 1 (Fig. 3A). To determine whether oligoribonucleo- tide synthesis per se is responsible for the slow extension, we bypassed the first nucleotide condensation step. Formation of the initial dinucleotide is thought to be the rate-limiting step in primer synthesis by all primases (18). Using a preformed AC dinucleotide, and CTP, we obtained similar maximal rates of primer use as in reactions primed de novo (Fig. 3A, Center). Likewise, bypassing primer synthesis altogether by supplying a preformed tetraribonucleotide, ACCC, resulted in the same maximum rate of extension (Fig. 3A, Right and Fig. S2). A summary of the results from these experiments is presented in Fig. 3B. The maximum observed rate of primer extension is identical, within error, for all three conditions. These results suggest that a step after primer synthesis, but before DNA synthesis, is critical for extension of primers by DNA poly- merase. The observed rate of primer extension increases line- arly with polymerase concentration until ∼400 nM polymerase, a twofold excess over the total gp4 hexamer present, after Fig. 1. Primer synthesis and extension by gp4 and T7 DNA polymerase. (A) gp4 unwinds dsDNA, using its C-terminal helicase domain. At PRSs, the gp4 primase domain synthesizes a short RNA, stabilizing it on the template and mediates its transfer to T7 DNA polymerase. (B) Gp4 enables T7 DNA polymerase to extend tetraribonucleotides; 0.1 μM gp4 hexamer or 0.2– 25 μM gp4 primase fragment (PF) was incubated with ssDNA in the absence or presence of T7 DNA polymerase for 5 min at 25 °C. Products are in- dicated to the right of the gel image. Pentamers are likely not extended efficiently (37, 38). (C)KlenowfragmentofE. coli DNA polymerase I and T4 DNA polymerase cannot extend short RNAs synthesized by gp4. Reactions were initiated by adding 10 mM MgCl2, and samples were taken at 10-s intervals. The 0 time point corresponds to a sample of the reaction before MgCl2 addition. Primer Synthesis Is More Efficient Than Primer Extension. We de- termined the kinetics of oligoribonucleotide synthesis and their extension by DNA polymerase using a 26-nt ssDNA template containing a single PRS. Using this template, full-length primers accumulate at a steady-state rate of ∼6.5 nM/s (Fig. 2A). This rate of primer accumulation corresponds to a kcat for primer synthesis − of 0.1 s 1 (calculated as described in SI Materials and Methods, using the initial rate of primer synthesis and data in Fig. S1). This result is inconsistent with a previous report (16) of the catalytic activity of a T7 primase fragment where synthesis of pppAC − occurs with a rate constant of ∼4s 1. Our result strongly sug- gests the existence of a step slower than dimer synthesis in the complete primer synthesis pathway. This step was not observ- able in the previous report because the authors used a substrate designed to produce dimers exclusively. Under our experimental conditions, accumulation of extended primer over time follows an exponential shape (Fig. 2B). Fitting extension product concentration over time to an exponential function yielded an observed rate constant for primer utilization Fig.
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