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In vitro reconstitution of DNA replication initiated by genetic recombination: a T4 bacteriophage model for a type of DNA synthesis important for all cells - PubMed Skip to main page content
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. 2019 Jan 1;30(1):146-159.
doi: 10.1091/mbc.E18-06-0386. Epub 2018 Nov 7.

In vitro reconstitution of DNA replication initiated by genetic recombination: a T4 bacteriophage model for a type of DNA synthesis important for all cells

Jack Barry  1 , Mei Lie Wong  1 , Bruce Alberts  1
Affiliations

In vitro reconstitution of DNA replication initiated by genetic recombination: a T4 bacteriophage model for a type of DNA synthesis important for all cells

Jack Barry et al. Mol Biol Cell. .

Abstract

Using a mixture of 10 purified DNA replication and DNA recombination proteins encoded by the bacteriophage T4 genome, plus two homologous DNA molecules, we have reconstituted the genetic recombination-initiated pathway that initiates DNA replication forks at late times of T4 bacteriophage infection. Inside the cell, this recombination-dependent replication (RDR) is needed to produce the long concatemeric T4 DNA molecules that serve as substrates for packaging the shorter, genome-sized viral DNA into phage heads. The five T4 proteins that catalyze DNA synthesis on the leading strand, plus the proteins required for lagging-strand DNA synthesis, are essential for the reaction, as are a special mediator protein (gp59) and a Rad51/RecA analogue (the T4 UvsX strand-exchange protein). Related forms of RDR are widespread in living organisms-for example, they play critical roles in the homologous recombination events that can restore broken ends of the DNA double helix, restart broken DNA replication forks, and cross over chromatids during meiosis in eukaryotes. Those processes are considerably more complex, and the results presented here should be informative for dissecting their detailed mechanisms.

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Figures

FIGURE 1:
FIGURE 1:
In the presence of UvsX protein, both gp59 and gp32 (32 protein) are needed to assemble a primosome on single-stranded DNA. RNA-primed DNA synthesis was carried out as described in Materials and Methods, either with (purple) or without (green) the addition of gp59. The single-stranded, circular M13 DNA template was incubated for 1 min at 37°C with the following DNA binding proteins: (A) gp32 (32 protein), (B) UvsX protein, and (C) both gp32 and UvsX proteins. DNA synthesis was then initiated by the addition of the T4 DNA polymerase holoenzyme and the T4 primosome (the DNA primase, gp61, plus the DNA helicase, gp41). When present, gp32 was present at 1.2 times the amount needed to cover all the single-stranded DNA (62 µg/ml), based on a binding site of 7 nucleotides per gp32 molecule (Jensen et al., 1976). The UvsX protein was present at 1.3 times the amount needed to cover all the single-stranded DNA (100 µg/ml), based on a binding site of five nucleotides per UvsX protein molecule. In (C) many DNA circles are covered by alternating patches of UvsX and gp32, each in a linear array that reflects each protein's cooperative DNA binding (Griffith and Formosa, 1985). In (D), we diagram the sequence of polynucleotide syntheses in these reactions—the primosome-catalyzed synthesis of a pentaribonucleotide (RNA primer) that then primes DNA synthesis by T4 DNA polymerase and its accessory proteins.
FIGURE 2:
FIGURE 2:
Schematic illustration of three possible types of recombination-dependent DNA synthesis. (Left) Conservative DNA synthesis of the type reported by Formosa and Alberts (1986), in which only a DNA single strand is produced. The dda protein was the T4 DNA helicase used in that earlier work; here we show that we can replace it with a mixture of gp41 DNA helicase and gp59 to drive the reaction shown on the left. (Right) Top, What happens when gp61 is added to form a complete primosome? If the T4 primosome (gp41 plus gp61) is loaded onto the displaced single-stranded tail, conservative DNA synthesis will produce a new DNA double helix in which both of the DNA strands in the new duplex are newly made. Bottom, If the T4 primosome is instead loaded inside the D-loop, DNA synthesis will occur semiconservatively. In that case, as at the standard replication fork, both of the two daughter DNA helices produced contain one old and one new strand.
FIGURE 3:
FIGURE 3:
Okazaki fragments are produced during recombination-dependent DNA synthesis only when both gp59 and the primosome are present. As diagrammed in Figure 2, the 1623-nucleotide-long single strand that primes recombination-dependent DNA synthesis is homologous to a region that begins 88 nucleotides from one end of a BglII linearized DNA template (which contains 7250 nucleotide pairs). DNA synthesis that is primed by the 3′OH end of the single strand can therefore extend this strand by 5539 nucleotides to the end of the double-stranded template, producing a 7162-nucleotide-long DNA single strand as product (7250 minus 88 nucleotides). In reactions 1 through 5 there is no functional primosome, and “snap-back” DNA synthesis (Goulian et al., 1968; Englund, 1971) on the 7-kb single-stranded DNA product is seen to produce small amounts of a hairpin helix formed from a DNA strand elongated to 14 kb (see Supplemental Figure S5A and Morrical et al., 1991). The addition of the 41 protein (gp41), a DNA helicase that moves in the 5′ to 3′ direction along a DNA single strand, stimulates snap-back synthesis; but it also produces a small amount of more slowly migrating DNA strands (henceforth designated as “19 kb”). The latter reaction, which is greatly increased by addition of gp59, is eliminated when the primosome is activated to produce Okazaki fragments on the single-stranded DNA (reaction 6). Standard reactions for recombination-dependent DNA synthesis were prepared as described in Materials and Methods. Linearized M13MP19 double-stranded DNA (prepared by BglII digestion) was preincubated with the DNA polymerase holoenzyme, gp32, the dda DNA helicase, and the recombination proteins UvsX and UvsY. The 61 protein (gp61 DNA primase), 41 protein (gp41, DNA helicase), and 59 protein (gp59) were present where indicated. After a brief equilibration at 37°C, recombination-dependent DNA synthesis was started by the addition of a linear single-stranded DNA molecule and the nucleotide substrates (see Materials and Methods). After different times at 37°C, the mixture was subjected to strong denaturing conditions and the radioactivity in the individual DNA strands produced, labeled by incorporation of [α-32P]dTTP at 830 Ci/mol, was analyzed by electrophoresis through a 0.8% agarose alkaline gel. An autoradiograph of the gel is shown at the (a) 10-min and (b) 20-min time periods.
FIGURE 4:
FIGURE 4:
Lagging-strand DNA synthesis creates a DNA topoisomerase requirement for recombination-dependent DNA synthesis on supercoiled, but not on linear DNA templates. (A) Amount of DNA synthesized when the double-stranded DNA template is supercoiled. (B) Amount of DNA synthesized under exactly the same conditions when the double-stranded DNA template is linear. (C) Alkaline agarose gel electrophoresis of the radioactive products from the reactions in A and B. Note that DNA products much longer than the supercoiled template molecule are formed in the absence of gp61 and topoisomerase (reaction 1), due to rolling-circle DNA synthesis from a small moving bubble (Formosa and Alberts, 1986). But when gp61 is added to complete the primosome, lagging-strand synthesis occurs on the supercoiled template. Now all of the products are short without topoisomerase present (reaction 2). When topoisomerase is added, long DNA products are synthesized on the supercoiled template like those in reaction 1, along with products resembling Okazaki fragments and a conspicuous band at 7.25 kb (reaction 3). In contrast, there is no effect of topoisomerase on the products made on the linear template in the presence of 61 protein (compare reactions 5 and 6). See Figure 5 for diagrams that illustrate these results. As described in Materials and Methods, M13MP19 double-stranded DNA, either supercoiled (form I) or BglII-linearized (form III), was preincubated with the DNA polymerase holoenzyme, gp32, gp41, gp59, UvsX, UvsY, and the dda DNA helicase. The gp61 DNA primase (61 protein) and the T4 DNA topoisomerase were also present where indicated. After temperature equilibration at 37°C, recombination-dependent DNA synthesis was started by the addition of the homologous 1623 nucleotide single-stranded DNA primer and the nucleotide substrates. After 7 and 14 min of DNA synthesis, the amount of product was determined from the incorporation of radioactively labeled [α-32P]dTTP precursor (panels A and B), and aliquots were analyzed by alkaline gel electrophoresis through 0.6% agarose followed by autoradiography (panel C).
FIGURE 5:
FIGURE 5:
Depiction of the products of recombination-dependent DNA synthesis on a supercoiled template. The products of reactions 1–3 in Figure 4 can be accounted for by the molecules shown here. For simplicity, supertwisted molecules are depicted in the covalently closed relaxed state. Because lagging-strand synthesis is semiconservative, the replication fork stalls when topoisomerase is not present to remove DNA winding strain ahead of the fork. The replication fork in the plus-topoisomerase reaction is depicted as unidirectional. Consequently, one of the form II circular products of this reaction contains a newly synthesized leading strand that is 7250 nucleotides long.
FIGURE 6:
FIGURE 6:
A major product of topoisomerase-dependent recombination-dependent DNA synthesis on the supercoiled M13MP19 (form I) template is a discrete band identified by electrophoresis on neutral gels as nicked M13MP19 (form II) double-stranded circles. Only those reactions with gp61 (61 protein) contain an active primosome. DNA synthesis on a supercoiled DNA template was carried out exactly as described for reactions 1–3 in Figure 4. Reactions were stopped with cold EDTA as described in Materials and Methods. Aliquots were then split and processed either for electron microscopy (see Figure 7) or gel electrophoresis. Shown here are autoradiographic analyses of the radioactive DNA products after electrophoresis on an alkaline 0.6% agarose gel (right) and a 0.8% agarose gel at neutral pH (left), following (a) 7 min and (b) 14 min of reaction. As seen previously, DNA synthesis in reaction 2 (no topoisomerase) is remarkably limited in comparison with the synthesis observed in the presence of topoisomerase (reaction 3). Most of the products of these DNA synthesis reactions move slowly in their native state on the neutral gel and are indistinguishable from one another. The exception is the prominent band in reaction 3, identified as the M13MP19 (form II) nicked circle by direct comparison with a randomly nicked M13MP19 DNA standard. Form II of M13MP19 is a predicted product of topoisomerase-dependent, semiconservative DNA synthesis (see Figure 5). The slowly moving products on the neutral gel include the DNA networks (“aggregates”) that form when UvsX, double-stranded DNA, and homologous single-stranded DNA react (see Formosa and Alberts, 1986). Also running slowly on the neutral gel are “rolling circle” molecules (molecules undergoing either strand displacement synthesis initiated from a nick or DNA synthesis from a small moving bubble), as well as positively supertwisted circles with a stalled replication bubble, whose unique structure apparently causes them to move slowly. (In contrast, simple circular molecules that are negatively or positively supertwisted run faster than nicked circles on the neutral gel.) Minor products detected in the neutral gel of size 1–5 kbp (reaction 3b) are thought to be single-stranded Okazaki fragments, displaced from their lagging strand template by “onion skin” DNA synthesis. The products made in reactions 2 and 3 are analyzed by electron microscopy in Figure 7.
FIGURE 7:
FIGURE 7:
Electron microscopy confirms and extends the conclusions derived from biochemical analyses. The DNA products present after 7 min of synthesis in the two reactions in Figure 6 containing gp61 and thus a complete primosome (reaction 2 without topoisomerase and reaction 3 with topoisomerase) were examined by electron microscopy. Aliquots from each reaction were spread on electron microscope grids (see Materials and Methods), and all visible molecules of each type were scored by sampling areas of equivalent size on each of six grids. In total, 362 molecules from reaction 2 (minus topoisomerase) and 619 molecules from reaction 3 (plus topoisomerase) could be characterized. Illustrated here are samples of each class of molecule observed to constitute more than 10% of the molecules in at least one of the two reactions, with the percentages indicated for both the “plus-topo” and “minus-topo” conditions. (A) Unreplicated negatively supercoiled templates, showing the naturally occurring variation in the amount of supercoiling observed. (B) Rolling circle, conservative DNA synthesis from a small moving bubble. (C) Two supertwisted DNA molecules that contain a stalled replication bubble; the positive supertwisting of the nonreplicated part of the circular template produces a variety of plectonemic supercoils. (D) Nicked monomer circle (form II), the majority product in the presence of DNA topoisomerase. (E) Rolling circle, semiconservative DNA replication from a nicked, form II template. (F) DNA tangle. (G) Histogram comparing the percentages observed for each of the six classes of product, with (+) and without (−) topoisomerase present. The most abundant minor products not illustrated here were linear DNA molecules of roughly template length; these constituted ∼10% of the total in both the plus-topo and minus-topo reactions and are thought to represent breakage products. Newly synapsed structures were detected in only minor amounts—as a circular double helix attached to a single-stranded, homologous primer that extends from a small D-loop (see Figure 8A). Scale bar indicates 100 nm.
FIGURE 8:
FIGURE 8:
Some effects of branch migration on a covalently closed, circular DNA template molecule containing a replication bubble stalled by superhelical tension. In the absence of a DNA topoisomerase, the initial product of recombination-dependent DNA synthesis on a circular template is a supertwisted molecule with a stalled replication bubble. An electron micrograph of such a molecule with supertwists removed is presented in A. This is the same as Molecule 1 in B, where the 3″ end of a homologous single-stranded DNA primer (blue) has been extended a few hundred nucleotides by leading-strand DNA synthesis, and an Okazaki fragment has been synthesized on the other side of the D-loop. As indicated, this molecule can undergo branch migration. In the top pathway, a slow UvsX-catalyzed branch migration occurs behind the stalled replication bubble, displacing the 3′OH end of the Okazaki fragment that had been inside the replication bubble, while leaving the 3′ OH end of the extended primer paired with its template (Molecule 2A). The newly extruded end of the Okazaki fragment then primes DNA synthesis to make the homologous DNA primer double-stranded, creating molecule 2B. A slow process of rolling-circle replication from a small moving bubble may subsequently occur, creating molecule 2C, in which Okazaki fragments that were synthesized inside the replication bubble have been repeatedly displaced by UvsX-driven branch migration. The product is a double helix that has been synthesized conservatively. In the bottom pathway, branch migration occurs at both ends of the stalled replication bubble, causing the 3′ OH end of the extended primer to be displaced from its template, stopping DNA synthesis (molecule 3). In the absence of topoisomerase, electron microscopy reveals numerous products that resemble the molecules diagrammed here (e.g., see Figure 7C and Supplemental Figure S4).
FIGURE 9:
FIGURE 9:
How cells use mediator proteins to control the outcome of recombination-dependent DNA replication (RDR). As described in the text, and observed in the experiments reported here, the loading of the gp41 DNA helicase on the displaced single strand is blocked by that strand's coating of UvsX protein, while being catalyzed at the front of the D-loop by the preferential binding of gp59 to the forked DNA and gp32 that is located there. As a result, the gp59 mediator protein loads gp41 only inside the D-loop. The gp41 then binds gp61 to produce semiconservative DNA replication. For the process of break-induced replication (BIR) thus far observed in S. cerevisiae and mammals, a different set of protein–protein interactions produces the opposite result, and DNA is replicated conservatively. Conservative DNA replication will be inherently error-prone, inasmuch as the mismatch repair systems that are essential for high replication fidelity cannot operate without the original parental strand to use as a reference.
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References

    1. Alberts BM. (1984). The DNA enzymology of protein machines. Cold Spring Harb Symp Quant Biol , 1–12. - PubMed
    1. Alberts BM. (1998). The cell as a collection of protein machines: preparing the next generation of molecular biologists. Cell , 291–294. - PubMed
    1. Alberts BM, Barry J, Bedinger P, Formosa T, Jongeneel CV, Kreuzer KN. (1983). Studies on DNA replication in the bacteriophage T4 in vitro system. Cold Spring Harb Symp Quant Biol , 655–668. - PubMed
    1. Alberts BM, Frey L. (1970). T4 bacteriophage gene 32: a structural protein in the replication and recombination of DNA. Nature , 1313–1318. - PubMed
    1. Ando RA, Morrical SW. (1998). Single-stranded DNA binding properties of the UvsX recombinase of bacteriophage T4: binding parameters and effects of nucleotides. J Mol Biol , 785–796. - PubMed

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