Volume 1, Issue 7, Pages 1001-1010 (June 1998) Coordinated Leading and Lagging Strand DNA Synthesis on a Minicircular Template  Joonsoo Lee, Paul D. Chastain, Takahiro Kusakabe, Jack D. Griffith, Charles C. Richardson  Molecular Cell  Volume 1, Issue 7, Pages 1001-1010 (June 1998) DOI: 10.1016/S1097-2765(00)80100-8

Figure 1 A Minicircle with a Replication Fork (a) Structure and nucleotide sequence of the minicircle. The duplex minicircle was constructed from chemically synthesized oligonucleotides as described in the Experimental Procedures. It consists of a dsDNA circle (70 bp) bearing a 5′-ssDNA tail (40 nt) to which the gp4 can bind. Two recognition sites (5′-TGGTC-3′) at which the gp4 primase can catalyze the synthesis of RNA primers are present on the lagging strand. One strand of the minicircle contains 33 cytosine residues but only 2 guanine residues, whereas the other contains 33 guanine residues and 2 cytosine residues. Consequently, leading and lagging strand synthesis can be identified and quantitated by measuring the incorporation of dGMP and dCMP, respectively. (b) DNA synthesis on the minicircle. DNA synthesis was carried out in the standard reaction containing minicircle, T7 DNA polymerase, T7 63 kDa gp4 helicase/primase, T7 gp2.5, 4 dNTP, ATP, and CTP as described in the Experimental Procedures. The reaction contains either [α-32P]dGTP or [α-32P]dCTP as indicated. The ratio of minicircle:gp4 hexamer:T7 DNA polymerase was 10:1:8. After incubation at 30°C for the indicated times (1 min, 2 min, and 3 min), the products of the reaction were denatured and analyzed by electrophoresis through 0.6% alkaline agarose gels. Molecular Cell 1998 1, 1001-1010DOI: (10.1016/S1097-2765(00)80100-8)

Figure 2 Gp2.5 Coordinates DNA Synthesis (a) Comparison of leading and lagging strand synthesis in the presence of gp2.5. DNA synthesis using the minicircle with the complete set of replication proteins described in the experiment presented in Figure 1b. Leading and lagging strand synthesis were measured by the incorporation of either [α-32P]dCMP or [α-32P] dGMP, respectively. After incubation at 30°C, aliquots (4 μl) from the reaction were assayed for the amount of radioactivity incorporated at the indicated times as described in the Experimental Procedures. (b) DNA synthesis in the absence of gp2.5. Reactions were as described in (a) except that gp2.5 was omitted from the reaction. (c) DNA synthesis in the presence of gp2.5Δ21C. Reactions were as described in (a) except that gp2.5Δ21C was replaced with wild-type gp2.5. Molecular Cell 1998 1, 1001-1010DOI: (10.1016/S1097-2765(00)80100-8)

Figure 5 Visualization of Minicircle Replication Products DNA replication reactions were carried out using the minicircle and T7 DNA polymerase, gp4, and gp2.5. Following incubation for 1 min at 30°C in the presence or absence of gp2.5, the samples were fixed with glutaraldehyde and directly prepared for EM including mounting on thin carbon foils, washing, and rotary shadowcasting with tungsten. (A) Product molecule synthesized in the presence of gp2.5. (B) Deproteinized product molecule in the presence of gp2.5. Following incubation for 1 min at 30°C in the presence of gp2.5, the DNA products were deproteinized and single-stranded segments complexed with E. coli SSB protein to generate thick extended segments as contrasted to the thin duplex DNA. The samples were then fixed and prepared for EM as in (A). (C) Product molecule synthesized in the absence of gp2.5. All electron micrographs were shown in reverse contrast. The bar is equivalent to 1000 bp. Molecular Cell 1998 1, 1001-1010DOI: (10.1016/S1097-2765(00)80100-8)

Figure 3 Effect of Inhibition of Lagging Strand Synthesis on Leading Strand Synthesis (a) Effect of ddCTP on leading strand synthesis in the absence of gp2.5. Leading strand DNA synthesis on the minicircle was measured by monitoring [α-32P]dGMP incorporation in a DNA synthesis reaction lacking gp2.5. ddCTP was present at a concentration of 3.75 μM (ddCTP:dCTP = 1:80). DNA synthesis over the indicated 5 min time period was assayed as described in the Experimental Procedures. (b) Effect of ddCTP on lagging strand synthesis. Lagging strand synthesis was measured by monitoring [α-32P]dCMP incorporation in a complete DNA synthesis reaction containing 3.75 μM ddCTP as described in (a) above. (c) Effect of ddCTP on leading strand DNA synthesis. Leading strand DNA synthesis in the complete DNA synthesis reaction containing 3.75 μM ddCTP was monitored as described in (a). (d) Effect of ddCTP on ongoing lagging strand synthesis. Lagging strand synthesis in the complete DNA synthesis reaction was measured as in (b). ddCTP was added to a concentration of 3.75 μM at 2.5 min of incubation. (e) Effect of ddCTP on ongoing leading strand synthesis. Leading strand DNA synthesis in the complete DNA synthesis was measured as in (c). ddCTP was added at 2.5 min of incubation as in (d). Molecular Cell 1998 1, 1001-1010DOI: (10.1016/S1097-2765(00)80100-8)

Figure 4 Processivity of Leading and Lagging Strand Synthesis (a) Scheme for measuring processivity and recycling of DNA polymerase. DNA synthesis catalyzed by the T7 replication system is initiated with the mutant F526 T7 DNA polymerase (dark) that discriminates against ddNTPs. A 10-fold excess of wild-type T7 DNA polymerase (light) that incorporates ddNTPs as well as dNTPs is added to the reaction. If either the leading or lagging strand F526 polymerase dissociates, it will be replaced by a wild-type polymerase. Consequently, if ddGTP or ddCTP are present during the reaction, leading or lagging strand synthesis, respectively, will be inhibited if a wild-type polymerase is able to incorporate the chain-terminating nucleotide. If the F526 polymerase dissociates upon completion of an Okazaki fragment, its replacement by a wild-type polymerase will lead to incorporation of ddCMP and inhibition. If the lagging strand polymerase recycles to a new primer, ddCMP cannot be incorporated. (b) Effect of ddGTP on leading strand synthesis. DNA synthesis on the minicircle was carried out with either the F526 polymerase, a 10-fold excess of wild-type polymerase, or a mixture of F526 polymerase and a 10-fold excess of wild-type polymerase. ddGTP (3.75 μM, ddGTP:dGTP = 1:80) was present during the reaction; leading strand synthesis was measured by the incorporation of [α-32P]dGMP. (c) Processivity of leading strand polymerase. DNA synthesis was initiated with the F526 polymerase and the reaction mixture was incubated for 5 min with or without the addition of a 10-fold excess of wild-type polymerase at 2.5 min; ddGTP was present as in (a). (d) Processivity of lagging strand synthesis. DNA synthesis was as in (c) except that ddCTP was replaced by ddGTP. Lagging strand synthesis was measured by the incorporation of [α-32P]dCMP. (e) Effect of ddGTP and ddCTP on leading strand synthesis. DNA synthesis was as in (c), except that either ddGTP or ddCTP were present during the reaction. Molecular Cell 1998 1, 1001-1010DOI: (10.1016/S1097-2765(00)80100-8)

Figure 6 Model for Coordinated Synthesis (a) DNA polymerase and gp4 hexamer initiate processive strand displacement synthesis. The ssDNA extruded by the helicase is coated with gp2.5. (b) T7 gp4 helicase rotates as it unwinds DNA, and its physical contact with gp2.5 spools the gp2.5-coated ssDNA (dark) into an ordered structure. (c) Upon reaching a critical mass, the gp2.5-coated ssDNA slows the helicase such that a primase site can be accessed by the primase domain of gp4. A growing Okazaki fragment creates a duplex segment that separates the original gp2.5-coated ssDNA from gp4. A second gp2.5-coated complex (light) now accumulates behind the helicase. (d) A replication loop is formed when the two complexes of gp2.5-coated ssDNA coalesce. As the reaction proceeds, the size of the new gp2.5-coated ssDNA complex (light) increases at the expense of the first (dark). (e) Upon completion of an Okazaki fragment, the lagging strand polymerase remains at the replication fork, perhaps through its association with gp2.5, and awaits the synthesis of a new primer, repeating the cycle. Molecular Cell 1998 1, 1001-1010DOI: (10.1016/S1097-2765(00)80100-8)