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 regulation 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 tetraribonucleotides 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 replication 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 25, 2021 − of ∼0.03 s 1 (Fig. 3A). To determine whether oligoribonucleotide 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 polymerase. 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 indicated 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 determined 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 suggests the existence of a step slower than dimer synthesis in the complete primer synthesis pathway. This step was not observable 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. 2. Primer utilization is slower than primer synthesis. (A, Left) Time − of ∼0.03 s 1, a value 10,000-fold smaller than the rate-limiting course of primer synthesis by gp4 using a ssDNA with a single PRS. Samples were taken before addition of MgCl (t = 0) and in 10-s intervals following its step of nucleotide incorporation, kpol, for T7 DNA polymerase 2 − (∼300 s 1) (17), but closer to the k for primer synthesis we addition. Products are indicated. (Right) Primer (tetra- and pentamer) con- cat centration vs. time. The initial rate of primer formation is shown. Product obtained. To extend this finding, we examined the relationship concentration was determined as described in Materials and Methods.(B, between primer synthesis by gp4 and primer extension by T7 Left) Time course of primer synthesis and extension by gp4 and T7 DNA DNA polymerase using a variety of DNA constructs (Fig. 2 C polymerase. Products are indicated (Ext = extended product). (Right) Product and D). In all cases, we observed that synthesis of oligonu- concentration vs. time. Extended primer (blue), free (unused) primer (red), cleotides by gp4 is more efficient than their utilization by and total primer (black). (C, Left) Time course of primer synthesis and ex- the polymerase. tension in the presence of gp4 and T7 DNA polymerase with a minicircle We tested the rate of primer utilization as a function of template, diagramed at Upper Right. Presence of gp4 and gp5/Trx is in- primer synthesis rate by varying the concentration of ATP and dicated. (Right) Product concentration vs. time for the minicircle reaction. (D, Left) Time course of primer synthesis and extension in the presence of CTP and measuring the rate of formation of extension product gp4 and T7 DNA polymerase with a fork template (diagramed Upper Right). (Fig. 3A). The observed primer utilization rate increases hyper- For comparison, a reaction using the minicircle in C as template is included

bolically as a function of nucleotide concentration. Fitting these (run on the same gel, but cropped for simplicity). (Right) Product concen- BIOCHEMISTRY data to a hyperbolic equation yielded a maximum utilization rate tration vs. time for the fork substrate.

Hernandez et al. PNAS | May 24, 2016 | vol. 113 | no. 21 | 5917 Downloaded by guest on September 25, 2021 should be the same as under multiple-turnover conditions. Primer extension under single-turnover primer synthesis proceeds with an − observed first-order rate constant of 0.03 s 1 (Fig. 4C), a rate identical to multiple-turnover primer extension experiments. Ex- amination of primer extension progress curves after short reaction times (<2 s) revealed the presence of a lag phase in primer extension (Fig. S3), suggesting that the kinetics we observe result from at least two steps in the pathway, of approximately equal magnitude (20, 21). Fitting the data by numerical integration to a model consisting of two consecutive, irreversible steps yields val- −1 −1 ues for rate constants, k1 = 0.1 s and k2 = 0.05 s . Intriguingly, the value of the first rate constant is identical to the kcat for full- length primer synthesis, which we show above to represent primer release. We interpret the second rate constant to represent a slow primer handoff to the polymerase, which may involve reposition- Fig. 3. Primer synthesis does not limit extension. (A, Left)Primerextension ing the primer/template into the polymerase active site. Therefore, reactions were carried out with varying concentrations of ATP and CTP, using at least two steps, of similar magnitude, occur on the initiation [α-32P] dGTP to visualize extension products only. Observed rate constants, pathway of Okazaki fragments. In parallel rapid-quench experi- kobs, for primer utilization were plotted as a function of nucleotide concen- ments, we determined that T7 DNA polymerase extends a primer tration. (Center) Observed rate constant for primer utilization as a function of annealed to a single-stranded DNA template rapidly (Fig. S4)in AC concentration. Reactions contained 0.5 mM CTP to allow extension of the agreement with previous reports (17). dinucleotide to a primer. (Right) Observed rate constant for primer extension as a function of the concentration of the tetraribonucleotide ACCC. (B)Tabular gp2.5 Enhances Primer Synthesis and Promotes Primer Extension. gp2.5 representation of the data from A.(C) Observed rate of primer utilization as a is critical for coordinating leading- and lagging-strand DNA syn- function of T7 DNA polymerase concentration. Reaction time courses were fit – as above, and the observed rate constant for extension is plotted as a function thesis (22 24). In addition to DNA binding (25), it interacts with of polymerase concentration.

whichitisconstant(Fig.3C), suggesting that inefficient primer extension is not a consequence of polymerase binding, which we have previously shown to be rapid (19).

Primer Release Is Rate Limiting as Determined by Pre–Steady-State Analysis. We examined the pre–steady-state kinetics of primer formation by gp4 to better understand the pathway of primer synthesis. Primer formation shows a pre–steady-state burst (Fig. 4A). This type of kinetic behavior is indicative of slow product release following its fast formation (20). The burst of primer formation − by gp4 occurs with an observed rate constant of ∼1.5 s 1,followed − by a steady state with a rate constant of 0.1 s 1. This result shows that primer synthesis occurs rapidly, and primers are retained by the enzyme after formation, only to be slowly released (Discussion). We conclude that the rate-limiting step in the full pathway for primer synthesis is not dinucleotide formation as previously suggested (6, 16, 18), but the release of primer from the primase active site. Primer formation is the result of three nucleotidyl transfer reactions. To obtain information on the rate of nucleotide incorporation for each step, as well as information about “abortive” release of intermediates, we used single-turnover primer synthesis experiments. A low concentration of labeled ATP is present with a high concentration of T7 gp4, template, and CTP. The experimental data fit well to a model with three nucleotide condensation steps (Fig. 4B). As reported previously (16), dinucleotide formation occurs rapidly, with a − rate constant of ∼4s 1. The dimer is in equilibrium with the enzyme, and readily dissociates, as evidenced from their accumulation in Fig. 4. Pre–steady-state analysis of primer synthesis and extension. (A)Primer primer synthesis reactions. Both subsequent CMP incorporation steps synthesis by gp4 occurs with a burst of primer formation. (Left)Timecourseof −1 are faster than dimer formation by at least fivefold (20 and >50 s primer synthesis by gp4 using an ssDNA with a single PRS using a rapid quench- for the second and third CMP incorporation steps, respectively). flow instrument. (leftmost well:10-min control). (Right) Primer formation vs. Although the trimer intermediate is capable of dissociating from the time. Data were fit to the pre–steady-state burst equation. (B)Single-turnover primase, the reaction is pushed toward tetramer formation by the primer synthesis by gp4. (Left) Time course of tetramer formation. (Right)Plot comparatively rapid sequential incorporation steps. of product formation vs. time. Data were fit by numerical integration to the model shown; solid lines show the fit. (C) Single-turnover primer synthesis and extension by gp4 and T7 DNA polymerase. (Left) Denaturing PAGE of reaction Primer Release and Handoff Are Control Points for DNA Chain time course. (Right) Plot of product formation vs. time. Used primer (blue), free Initiation. If primer release is rate limiting in initiating lagging- primer (red), and total primer (black). Data were fit to single- or double- strand DNA synthesis, the rate of primer utilization under con- exponential functions. Observed rate constants and reaction amplitudes are ditions where primer synthesis occurs in a single-turnover regime indicated.

5918 | www.pnas.org/cgi/doi/10.1073/pnas.1604894113 Hernandez et al. Downloaded by guest on September 25, 2021 T7 DNA polymerase and gp4 through its acidic C-terminal tail acidic C-terminal tail that retains DNA binding but does not (26, 27). However, the effect of gp2.5 on primer synthesis by gp4 interact with other T7 replication proteins (30) (Fig. 5B and has thus far been equivocal (28, 29). Fig. S5C). Importantly, the presence of gp2.5 does not inhibit Gp2.5 slightly tempers primer synthesis using a single-PRS ssDNA DNA synthesis by T7 DNA polymerase. (Fig. S7). Fitting the template (6.5 vs. 3 nM/s, − and + gp2.5, respectively). DNA binding appearance of extension products over time to an exponential by gp2.5 may interfere with the ability of gp4 to engage the PRS. We function yields an observed rate constant for primer extension − note, however, that the proportion of dimer intermediate in the of 0.1 s 1. This value is in line with the value for the observed presence of gp2.5 is diminished by approximately fourfold (Fig. 5A). rate constant for primer release from gp4. We also examined We observed a similar result with a fork substrate, which has to be the effect of gp2.5 on primer extension under single-turnover first unwound to reveal the PRS (Fig. S5B). Decreased dimer ac- primer synthesis conditions. In agreement with the results in cumulation in the presence of gp2.5 suggests that it may inhibit Fig. 5B, gp2.5 enhances the rate of formation of extension dissociation of primer intermediates from gp4. Competition experi- product, but limits overall extension (Fig. 5C and Fig. S5A). ments, where increasing concentrations of AC dinucleotide were Rate constants for primer extension in the presence of gp2.5 in titrated into primer synthesis reactions with or without gp2.5, suggest multiple-and single-turnover primer synthesis conditions are that gp2.5 decreases dimer dissociation modestly (Fig. S6). identical, within error. The effect of gp2.5 on extension of gp4-synthesized primers These results suggest that two observable kinetic steps, of ap- by T7 DNA polymerase is complex. Gp2.5 inhibits primer ex- proximately equal magnitude, are rate limiting for DNA initiation tension (Fig. S5A),whichmaybeduetoDNAbindingbygp2.5 when only gp4 and T7 DNA polymerase are present. We surmise sequestering available template. However, extension products that primer release and handoff are these critical steps. In the are detected at earlier times, reaching a plateau earlier than presence of gp2.5, however, the extension rate constant is equal to reactions lacking gp2.5 (Fig. 5B), or those that contain either the primer release rate, suggesting that gp2.5 enables optimal E. coli SSB protein or gp2.5 Δ-C, a gp2.5 variant lacking the assembly of the priming complex and/or primer positioning into the polymerase active site. Consequently, the only step-limiting primer extension in the presence of all T7 replisome components is the release of full-length primer from gp4.

gp2.5 Regulates Lagging-Strand Synthesis by Limiting Primer Synthesis and Utilization. One of the hallmarks of coordinated DNA synthesis is the relatively narrow size range of Okazaki fragments, centering around1,000ntinlengthinT7-infectedE. coli, and under coordinated DNA synthesis in vitro (22, 23), whereas fragments resulting from uncoordinated synthesis are 500 nt or smaller. This observation suggests that during uncoordinated synthesis, primer extension and/or Okazaki fragment termination occur frequently and that coordination of DNA synthesis involves regulatory timing events on the lagging strand. We reasoned that gp2.5 leads to longer Okazaki fragments by limiting primer extension events. We therefore examined the effect of gp2.5 and gp2.5 Δ-C on Okazaki fragments derived from a minicircle template in reactions with radiolabeled CTP. This labeling results in primers and Okazaki fragments of equal specific activity, allowing a more accurate comparison. Lagging-strand products in reactions containing WT gp2.5 display a higher molecular weight than in reactions lacking gp2.5 or in the presence of gp2.5 Δ-C. However, our ability to accurately determine the size of the products was precluded by our use of native conditions during electrophoresis to maintain the integrity of the labeled RNA termini (Fig. 6A). The presence of gp2.5 leads to decreased levels of extended primer (Fig. 6B). In the presence of gp2.5, ∼4% of primers are used, however, extension products in reactions containing gp2.5 reach a higher molecular weight, greater than 1.5 kb (close to the mobility limit of the gel), determined by denaturing PAGE, and are evident even at the shortest time point analyzed (10 s) Fig. 5. gp2.5 increases the efficiency of primer synthesis and extension (Fig. 6C). The periodicity in the pattern of extension products also while restricting product formation. (A) Effect of gp2.5 on primer synthesis. shows that PRSs are over-used, i.e., intervening bands representing (Upper) Primer synthesis ± 3 μM gp2.5 (t = 0, 10, 20, 30, 40, and 50 s). (Lower) − + μ = initiation/termination events of primer extension are overrepresented primer formation vs time. (blue) and (red) 3 M gp2.5 (t 0, 10, 20, 30, Δ 40, 50, and 60 s). The initial rate of primer formation is indicated. (B) Effect in the absence of gp2.5 and in the presence of gp2.5 -C. However, of gp2.5 on primer synthesis and extension. (Upper) Primer synthesis and when WT gp2.5 is present, these intervening bands are greatly extension in the absence and presence of gp2.5. Primer and extension reduced. Thus, gp2.5 restricts primer utilization, perhaps by products are indicated (t = 0, 10, 20, 30, 40, 50, and 60 s). (Lower) normalized preventing interactions leading to priming complex formation, product formation vs. time for reactions lacking gp2.5 (blue), or with WT resulting in bypass of PRSs in the template. This bypass mech- (red) or Δ-C gp2.5 (green). (t = 0, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, and anism may allow leading- and lagging-strand DNA synthesis to 100 s). Observed rate constants are indicated. (C) Effect of gp2.5 on single- proceed at comparable rates. turnover primer synthesis and extension. (Left) Primer synthesis and exten- ± μ = sion (Materials and Methods) 20 M gp2.5. (t 0, 5, 10, 20, 30, 40, 50, 60, Discussion 70, 80, 90, 100, and 300 s). Primer and extension products are indicated. Right, normalized product formation vs. time for single-turnover reactions Replication of the lagging strand is a complex process that requires

lacking gp2.5 (blue), or in the presence of WT gp2.5 (red). (t = 0, 5, 10, 20, 30, a synergy between DNA primase, DNA helicase, DNA polymerase, BIOCHEMISTRY 40, 50, 60, 70, 80, 90, and 100 s). Observed rate constants are indicated. and single-stranded DNA binding protein. In addition, replication

Hernandez et al. PNAS | May 24, 2016 | vol. 113 | no. 21 | 5919 Downloaded by guest on September 25, 2021 attribute to inefficient positioning/engagement of the primer/ template by the polymerase. In the presence of gp2.5, however, primer handoff ceases to be rate limiting, probably due to efficient formation of an extension-competent complex between gp4, DNA polymerase, and primer/template. Once the primer/ template is secured by the polymerase and correctly positioned, DNA synthesis proceeds rapidly. Our data provide a model for regulating Okazaki fragment length through limiting primer utilization (Fig. 7B). Gp4, trans- locating in a 5′–3′ direction, encounters a PRS and synthesizes a primer, pausing the replisome while the primer remains bound to gp4 (33–35) before its transfer to DNA polymerase. In this step, gp2.5 promotes more efficient full-length primer synthesis. The primer is handed off to T7 DNA polymerase, a slow event in the absence of gp2.5, but fast in its presence. gp2.5 likely en- forces a binding orientation that enables efficient positioning of the primer on the polymerase active site. The primer is rapidly extended by T7 DNA polymerase. As the fork progresses, gp4

Fig. 6. Gp2.5 regulates Okazaki fragment length through primer utilization. (A) Effect of WT or Δ-C gp2.5 (3 μM) on lagging-strand DNA (labeled at the primer terminus). (Left) Samples quenched at 1 or 5 min were loaded on a 0.8% agarose gel (size markers are indicated). Brackets indicate range of extension products. (Right) Effect of gp2.5 on primer synthesis and extension using a minicircle. Samples taken at 10-s intervals and loaded on a denaturing 25% polyacrylamide gel. (B) Quantification of A.(C) Effect of gp2.5 on the time course of lagging-strand product formation. Samples were loaded on a 5% denaturing polyacrylamide gel; 5′ end-labeled size markers are indicated. A labeling artifact dependent on gp5 and gp2.5 is indicated (*).

of the lagging strand must be coordinated with events on the leading strand. Although great advances have been made in identifying key enzymes and steps in DNA replication, precisely how the interactions of these components are regulated at the replication fork is largely unknown. We propose a kinetic model for the initiation of Okazaki fragments by the T7 replisome (Fig. 7A). After encountering a PRS, the primase domain of gp4 catalyzes the formation of pppAC with a rate − constant of 4 s 1. This dinucleotide intermediate is in equilibrium with the enzyme, most likely explaining why the dimer is prominent in primer synthesis reactions. The dimer is extended by two rapid, consecutive NMP additions to form a tetramer. Although dissociation of trimer from gp4 is possible, its fast extension to a tetramer prevents its accumulation in the reaction. Release of tetramer from − the primase active site is slow, with a rate of release of 0.1 s 1.The − rate of association of tetramer to gp4 is very slow, 5 × 10 3 μM/s (calculated from the Ki of preformed tetramer on primer synthesis and the release rate; Fig. S8), making dissociation of primer from Fig. 7. Key steps in lagging-strand initiation by gp4 and T7 DNA polymerase the primase domain of gp4 essentially irreversible. The molecular and the regulation of Okazaki fragment length by gp2.5. (A) Primers are basis for this step, which we hypothesize involves a conformational rapidly synthesized by gp4, but their release is slow, as is their handoff to rearrangement in the primase, is under investigation. Slow release polymerase. Gp2.5 lowers the rate of primer synthesis but promotes for- from primase may allow efficient positioning of the polymerase or mation of full-length primer by gp4 and assists in assembly of an extension- provide time for other events regulating coordination. competent complex. After the primer/template is correctly engaged by the After synthesis, the tetraribonucleotide bound to gp4 and the polymerase, DNA synthesis proceeds rapidly. (B) Gp2.5 regulates Okazaki template, is transferred to the DNA polymerase active site for fragment length by restricting access to PRSs and by limiting primer exten- extension. Our previous work suggests that DNA polymerase sion. In the absence of gp2.5, encounter of each (or many) PRS leads to binding to gp4 occurs rapidly (19, 31). In addition, complex primer synthesis, and release of the Okazaki fragment by the polymerase results in short Okazaki fragments. In the presence of gp2.5, most newly formation is not sufficient for primer extension (32), suggesting encountered PRSs are bypassed, leading to less frequent priming and longer thattherateweobserveisdirectly related to the formation of Okazaki fragments. Ultimately, dissociation of gp2.5 from the template, or an extension-competent complex. In the absence of gp2.5, the from the lagging-strand polymerase, occurs and a primer is extended at a measured primer extension rate constant is slow, which we new recognition site, releasing the nascent Okazaki fragment.

5920 | www.pnas.org/cgi/doi/10.1073/pnas.1604894113 Hernandez et al. Downloaded by guest on September 25, 2021 encounters additional PRSs. The presence of gp2.5 either masks Materials and Methods their recognition or if primer synthesis occurs, formation of the Details of experimental methods are shown in SI Materials and Methods. priming complex is prevented. In either case, the lagging-strand polymerase continues extension unimpeded. In the absence of Protein Expression and Purification. Proteins were purified as previously de- gp2.5, however, when gp4 encounters a PRS, it begins synthesis. scribed (12, 19, 36), with a slight modification in the case of gp4 detailed in SI This event retards the lagging-strand polymerase and triggers its Materials and Methods. release (35) and the eventual reformation of a priming complex, Primer Synthesis Assays. Reactions (22) were initiated by addition of a final leading to a short Okazaki fragment. A high local concentration concentration of 10 mM MgCl2 and incubated at 25 °C. of primer synthesis products could trigger disengagement of the lagging-strand polymerase. The interplay between DNA synthe- Data Analysis. Product progress curves were fit using Kaleidagraph (Synergy) sis rate and dissociation kinetics of gp2.5 from DNA, or another or Kintek Explorer (Kintek). replisome component may lead to sporadic unmasking of PRSs. Such a scenario would allow initiation of primer synthesis and ACKNOWLEDGMENTS. We thank C. Cifuentes-Rojas and C.C.R. laboratory members for comments, B. Zhu for WT gp2.5, and S. Moskowitz for figure formation of a priming complex, leading to the lagging-strand preparation. This work was supported by National Institutes of Health polymerase dissociating from the Okazaki fragment. Grants F32GM101761 (to A.J.H.) and GM54397 (to C.C.R.).

1. Kornberg A, Baker TA (1992) DNA Replication (W.H. Freeman, New York), 2nd Ed, 24. Kim YT, Tabor S, Bortner C, Griffith JD, Richardson CC (1992) Purification and char- pxiv. acterization of the bacteriophage T7 gene 2.5 protein. A single-stranded DNA- 2. Costa A, Hood IV, Berger JM (2013) Mechanisms for initiating cellular DNA replication. binding protein. J Biol Chem 267(21):15022–15031. Annu Rev Biochem 82:25–54. 25. Hyland EM, Rezende LF, Richardson CC (2003) The DNA binding domain of the gene 3. Balakrishnan L, Bambara RA (2013) Okazaki fragment metabolism. Cold Spring Harb 2.5 single-stranded DNA-binding protein of bacteriophage T7. J Biol Chem 278(9): Perspect Biol 5(2):a010173. 7247–7256. 4. Hamdan SM, Richardson CC (2009) Motors, switches, and contacts in the replisome. 26. Ghosh S, Hamdan SM, Richardson CC (2010) Two modes of interaction of the single- – Annu Rev Biochem 78:205 243. stranded DNA-binding protein of bacteriophage T7 with the DNA polymerase- 5. Lee SJ, Richardson CC (2011) Choreography of bacteriophage T7 DNA replication. Curr thioredoxin complex. JBiolChem285(23):18103–18112. – Opin Chem Biol 15(5):580 586. 27. Kim YT, Tabor S, Churchich JE, Richardson CC (1992) Interactions of gene 2.5 protein 6. Frick DN, Richardson CC (2001) DNA primases. Annu Rev Biochem 70:39–80. and DNA polymerase of bacteriophage T7. J Biol Chem 267(21):15032–15040. 7. Mendelman LV, Notarnicola SM, Richardson CC (1992) Roles of bacteriophage T7 28. Mendelman LV, Richardson CC (1991) Requirements for primer synthesis by bacte- gene 4 proteins in providing primase and helicase functions in vivo. Proc Natl Acad Sci riophage T7 63-kDa gene 4 protein. Roles of template sequence and T7 56-kDa gene 4 USA 89(22):10638–10642. protein. J Biol Chem 266(34):23240–23250. 8. Qimron U, Lee SJ, Hamdan SM, Richardson CC (2006) Primer initiation and extension 29. He ZG, Richardson CC (2004) Effect of single-stranded DNA-binding proteins on the by T7 DNA primase. EMBO J 25(10):2199–2208. helicase and primase activities of the bacteriophage T7 gene 4 protein. J Biol Chem 9. Kato M, Ito T, Wagner G, Richardson CC, Ellenberger T (2003) Modular architecture of – the bacteriophage T7 primase couples RNA primer synthesis to DNA synthesis. Mol 279(21):22190 22197. Cell 11(5):1349–1360. 30. Marintcheva B, Marintchev A, Wagner G, Richardson CC (2008) Acidic C-terminal tail 10. Kusakabe T, Richardson CC (1997) Gene 4 DNA primase of bacteriophage T7 mediates of the ssDNA-binding protein of bacteriophage T7 and ssDNA compete for the same – the annealing and extension of ribo-oligonucleotides at primase recognition sites. binding surface. Proc Natl Acad Sci USA 105(6):1855 1860. J Biol Chem 272(19):12446–12453. 31. Hamdan SM, et al. (2007) Dynamic DNA helicase-DNA polymerase interactions assure 11. Nakai H, Richardson CC (1986) Dissection of RNA-primed DNA synthesis catalyzed by processive replication fork movement. Mol Cell 27(4):539–549. gene 4 protein and DNA polymerase of bacteriophage T7. Coupling of RNA primer 32. Wallen JR, Majka J, Ellenberger T (2013) Discrete interactions between bacteriophage and DNA synthesis. J Biol Chem 261(32):15217–15224. T7 primase-helicase and DNA polymerase drive the formation of a priming complex 12. Lee SJ, Zhu B, Hamdan SM, Richardson CC (2010) Mechanism of sequence-specific containing two copies of DNA polymerase. Biochemistry 52(23):4026–4036. template binding by the DNA primase of bacteriophage T7. Nucleic Acids Res 38(13): 33. Pandey M, et al. (2009) Coordinating DNA replication by means of priming loop and 4372–4383. differential synthesis rate. Nature 462(7275):940–943. 13. Frick DN, Baradaran K, Richardson CC (1998) An N-terminal fragment of the gene 4 34. Lee JB, et al. (2006) DNA primase acts as a molecular brake in DNA replication. Nature helicase/primase of bacteriophage T7 retains primase activity in the absence of 439(7076):621–624. helicase activity. Proc Natl Acad Sci USA 95(14):7957–7962. 35. Hamdan SM, Loparo JJ, Takahashi M, Richardson CC, van Oijen AM (2009) Dynamics 14. Kato M, Ito T, Wagner G, Ellenberger T (2004) A molecular handoff between bacte- of DNA replication loops reveal temporal control of lagging-strand synthesis. Nature riophage T7 DNA primase and T7 DNA polymerase initiates DNA synthesis. J Biol 457(7227):336–339. Chem 279(29):30554–30562. 36. Lee SJ, Richardson CC (2010) Molecular basis for recognition of nucleoside tri- 15. Nakai H, Richardson CC (1986) Interactions of the DNA polymerase and gene 4 protein phosphate by gene 4 helicase of bacteriophage T7. J Biol Chem 285(41):31462–31471. of bacteriophage T7. Protein-protein and protein-DNA interactions involved in RNA- 37. Romano LJ, Richardson CC (1979) Characterization of the ribonucleic acid primers and – primed DNA synthesis. J Biol Chem 261(32):15208 15216. the deoxyribonucleic acid product synthesized by the DNA polymerase and gene 16. Frick DN, Kumar S, Richardson CC (1999) Interaction of ribonucleoside triphosphates 4 protein of bacteriophage T7. J Biol Chem 254(20):10483–10489. – with the gene 4 primase of bacteriophage T7. J Biol Chem 274(50):35899 35907. 38. Tabor S, Richardson CC (1981) Template recognition sequence for RNA primer syn- 17. Patel SS, Wong I, Johnson KA (1991) Pre-steady-state kinetic analysis of processive thesis by gene 4 protein of bacteriophage T7. Proc Natl Acad Sci USA 78(1):205–209. DNA replication including complete characterization of an exonuclease-deficient 39. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram mutant. Biochemistry 30(2):511–525. quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 18. Kuchta RD, Stengel G (2010) Mechanism and evolution of DNA primases. Biochim 248–254. Biophys Acta 1804(5):1180–1189. 40. Tabor S, Huber HE, Richardson CC (1987) Escherichia coli thioredoxin confers processivity 19. Hamdan SM, et al. (2005) A unique loop in T7 DNA polymerase mediates the binding on the DNA polymerase activity of the gene 5 protein of bacteriophage T7. JBiolChem of helicase-primase, DNA binding protein, and processivity factor. Proc Natl Acad Sci – USA 102(14):5096–5101. 262(33):16212 16223. 20. Johnson KA (2013) A century of enzyme kinetic analysis, 1913 to 2013. FEBS Lett 41. Cao W, De La Cruz EM (2013) Quantitative full time course analysis of nonlinear 587(17):2753–2766. enzyme cycling kinetics. Sci Rep 3:2658. 21. Kuzmic P (2009) Application of the Van Slyke-Cullen irreversible mechanism in the 42. Wang MH, Zhao KY (1997) A simple method for determining kinetic constants of analysis of enzymatic progress curves. Anal Biochem 394(2):287–289. complexing inactivation at identical enzyme and inhibitor concentrations. FEBS Lett 22. Lee J, Chastain PD, 2nd, Kusakabe T, Griffith JD, Richardson CC (1998) Coordinated 412(3):425–428. leading and lagging strand DNA synthesis on a minicircular template. Mol Cell 1(7): 43. Johnson KA (2009) Fitting enzyme kinetic data with KinTek Global Kinetic Explorer. 1001–1010. Methods Enzymol 467:601–626. 23. Lee J, Chastain PD, 2nd, Griffith JD, Richardson CC (2002) Lagging strand synthesis 44. Cheng Y, Prusoff WH (1973) Relationship between the inhibition constant (K1) and in coordinated DNA synthesis by bacteriophage t7 replication proteins. J Mol Biol the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzy- 316(1):19–34. matic reaction. Biochem Pharmacol 22(23):3099–3108. BIOCHEMISTRY

Hernandez et al. PNAS | May 24, 2016 | vol. 113 | no. 21 | 5921 Downloaded by guest on September 25, 2021