Exchange of DNA polymerases at the replication fork of bacteriophage T7

Abstract

T7 gene 5 DNA polymerase (gp5) and its processivity factor, Escherichia coli thioredoxin, together with the T7 gene 4 DNA helicase, catalyze strand displacement synthesis on duplex DNA processively (>17,000 nucleotides per binding event). The processive DNA synthesis is resistant to the addition of a DNA trap. However, when the polymerase-thioredoxin complex actively synthesizing DNA is challenged with excess DNA polymerase-thioredoxin exchange occurs readily. The exchange can be monitored by the use of a genetically altered T7 DNA polymerase (gp5-Y526F) in which tyrosine-526 is replaced with phenylalanine. DNA synthesis catalyzed by gp5-Y526F is resistant to inhibition by chain-terminating dideoxynucleotides because gp5-Y526F is deficient in the incorporation of these analogs relative to the wild-type enzyme. The exchange also occurs during coordinated DNA synthesis in which leading- and lagging-strand synthesis occur at the same rate. On ssDNA templates with the T7 DNA polymerase alone, such exchange is not evident, suggesting that free polymerase is first recruited to the replisome by means of T7 gene 4 helicase. The ability to exchange DNA polymerases within the replisome without affecting processivity provides advantages for fidelity as well as the cycling of the polymerase from a completed Okazaki fragment to a new primer on the lagging strand.

Fig. 1.

Model of the bacteriophage T7 replication fork. The T7 replisome consists of the DNA polymerase (gp5), the processivity factor E. coli thioredoxin (trx), the primase/helicase (gp4), and the ssDNA-binding protein (gp2.5). The gp5/trx complex synthesizes the leading strand continuously as the helicase unwinds the duplex. The lagging strand is synthesized as short Okazaki fragments. Synthesis of the lagging strand is initiated from RNA primers (green) catalyzed by the primase domain of gp4. A loop is formed on the lagging strand to align both gp5/trx complexes. Gp2.5 coats the ssDNA regions of the lagging strand generated as the helicase unwinds the DNA.

Fig. 2.

Polymerase activity of gp5/trx and gp5-Y526F/trx on M13 ss- and dsDNA. DNA synthesis by gp5/trx (Upper) and gp5-Y526F/trx (Lower) was monitored by the amount of [³H]dTMP incorporated into DNA over time as described in Materials and Methods. (A) DNA synthesis on primed M13 ssDNA was carried out in the presence and absence of ddGTP. (B) Strand displacement DNA synthesis catalyzed by the T7 DNA polymerase in the presence of gene 4 helicase on M13 dsDNA was carried out in the presence (filled circles) or absence (open circles) of ddGTP.

Fig. 3.

Exchange of DNA polymerases during DNA synthesis on ssDNA templates. DNA polymerase exchange assays were carried out on primed M13 ssDNA in the absence (A) or presence (B) of E. coli SSB protein (165 μg/40-μl reaction) as described in Materials and Methods. Reactions were initiated by the addition of 10 nM gp5-Y526F/trx at 30°C, and a 10-fold molar excess (100 nM) gp5/trx was added to the reaction 30 s later. Reaction mixtures were performed in the absence (open circles) or presence (filled circles) of 50 μM ddGTP.

Fig. 4.

Processivity of gp5/trx and gp4 on dsDNA. (A) The M13 dsDNA or (B) minicircle DNA template was preincubated with gp5/trx and gp4 as described in Materials and Methods. Reactions were initiated with MgCl₂, dNTPs and [α-³³P]dATP in the absence or presence of (dC)₂₀₀·oligo(dG)₂₀ to trap any free enzyme that dissociates from the circular DNA. Aliquots were removed from the reaction at the indicated times, and DNA synthesis was stopped by the addition of EDTA before separation of the products on a 0.8% agarose gel. In the control experiment, the trap was added during the preincubation step.

Fig. 5.

Exchange of DNA polymerases during strand displacement synthesis. (A) Exchange reactions were performed as described in Materials and Methods in the presence of 2 nM M13 dsDNA. Reactions were initiated by the addition of 16 nM gp4, 4 μM gp2.5, and 16 nM gp5-Y526F/trx. At 1 min, a 10-fold excess of WT gp5/trx was added to the reaction followed by an addition of 50 μM ddGTP at 2 min (red). At the indicated times, aliquots were removed from the assay to determine the amount of [³H]dTMP incorporated into the DNA. In the gp5-Y526F/trx control reactions (black), DNA synthesis was monitored in the absence of excess WT gp5/trx but in the presence and absence of ddGTP. In a WT gp5/trx control reaction (blue), a 10-fold excess of WT gp5/trx was added to the reaction without the addition of ddGTP. (B) In the reverse scheme, 16 nM gp5/trx was used to initiate the reaction, and a 10-fold excess of gp5-Y526F/trx was then added at 1 min and ddGTP at 2 min.

Fig. 6.

Polymerase exchanges during coordinated DNA synthesis. (A) Polymerase exchanges during leading-strand synthesis. Reactions using a 70-nucleotide minicircle and primer–template were carried out as described in Materials and Methods. Reactions were initiated on 16 nM minicircle by the addition of 2 nM hexameric gp4, 1 μM gp2.5, and 14 nM gp5-Y526F/trx. At 1 min, a 10-fold excess of WT gp5/trx was added to the reaction followed by an addition of 50 μM ddGTP at 2 min (red). At the indicated times, aliquots were removed from the assay to determine the amount of [³H]dGMP incorporated into the DNA. In gp5-Y526F/trx control reactions (black), DNA synthesis was monitored in the absence of excess WT gp5/trx but with the presence and absence of ddGTP. In a WT gp5/trx control reaction (blue), a 10-fold excess of WT gp5/trx was added to the reaction without adding ddGTP. (B) Exchange reactions during coordinated leading- and lagging-strand syntheses. Reactions were identical to those described above except ATP and CTP were present to allow for lagging-strand synthesis.