Replication of duplex DNA of phage phi X174 reconstituted with purified enzymes.

Abstract

Replication of the covalently closed duplex replicative form (RF) of phage gX174 DNA has been achieved by couplin two known enzyme systems: (i) synthesis of viral strand circles (SS) from RF, and (if) conversion of SS to nearly complete RF (RF II. In this coupled system, activated RF (gene A-RF II complex) was a more efficient template and generated as many as 10 RF II molecules per RF input, at a rate commensurate with SS synthesis. The (12,13). One system (RF --SS) requires the participation of OX-encoded gene A protein, host rep protein, SSB, and DNA polymerase III holoenzyme to produce multiple copies of covalently closed, single-stranded viral circles from RF I. Replication is initiated by gene A protein, which creates a nick in the viral strand at the origin of RF replication (14-16). Synthesis by holoenzyme of viral strands, facilitated by helicase action of rep protein (17), proceeds by a looped rolling-circle mechanism (13).The second system needed for RF synthesis is the initial reaction of complementary strand synthesis (SS --RF); the nascent viral strands now provide the template for complementary strand synthesis. Thus, viral and complementary strands are synthesized by distinct mechanisms (5, 14), analogous to the continuous and discontinuous mechanisms proposed in host chromosome synthesis (7,14). Although synthetic viral strands isolated from the RF --SS reaction were shown to serve as templates for the conversion of SS to RF (18), the reagents for the latter reaction strongly inhibited the RF --SS reaction. With more extensive purification of the numerous proteins required for both of these stages, coupling of the SS -RF with the RF SS reactions has been achieved and is the subject of this report. Operation of the coupled system now provides an opportunity to investigate basic questions about the mechanisms of viral and complementary strand synthesis in RF duplication. MATERIALS AND METHODS MaterialsBuffer A is 0.1 M Tris-HCI (pH 7.5), 20% (wt/vol) sucrose, 40 mM dithiothreitol, and bovine serum albumin at 0.2 mg/ml; buffer B is 0.1 M Tris-HCl (pH 8.1), 50 mM MgCl2, 5 mM dithiothreitol, bovine serum albumin at 0.5 mg/ml, and 50% (vol/vol) glycerol; buffer C is 0.02 M Tris-HCI (pH 8.1), 1 M NaCl, 5 mM EDTA, bovine serum albumin at 0.1 mg/ml, 1 mM dithiothreitol, and 10% (vol/vol) glycerol.Extensively purified E. coli replication proteins were: rep protein (4 X 107 units/mg) (19), gene A protein* (5.5 X 106 units/mg) (20), and dUTPase (2 X 107 units/mg) (18). The other replication proteins were as described (21). RNA polymerase holoenzyme (22)

The E75 ecdysone-inducible gene responsible for the 75B early puff in Drosophila encodes two new members of the steroid receptor superfamily.

“…), containing the (bX174 origin of replication cloned in between the HindIII and BamHI sites of pBR322. This allowed the synthesis of single-stranded probe DNA (Arai et al 1980), which was performed by the incubation of supercoiled plasmid DNA with gene A protein, rep and ssb proteins, and DNA polymerase III holoenzyme (all generously provided by the A. Kornberg laboratory) in a reaction containing 20 mM Tris-C1 (pH 7.5), 80 ~g/ml BSA, 4% glycerol, 20 mM DTT, 1 mM ATP, 16 mM concentrations of the three unlabeled deoxynucleotides and 1.6 mM concentrations of the labeled deoxynucleotide for 1 hr at 30°C. EDTA was then added to 20 mM, SDS to 0.1%, and proteinase K to 50 ~tg/ml.…”

DNA replication of single-stranded Escherichia coli DNA phages

“…Primosome-associated ~X RFI or ~,X RFIV DNA is a much better substrate for cleavage with gene A protein than superhelical RFI DNA, as shown by the 20-fold enhancement of the initial rate of DNA synthesis [35]. Besides facilitation of the RF DNA nicking by gene A protein, the primosome may also serve as a helicase during RF DNA replication and may initiate in the replication fork priming of complementary strand synthesis [24,58,59[. Conservation of the primosome on the RF molecule containing the original complementary strand, and thereby favouring its replication within the cell, may explain the dominant role of the genotype of the parental complementary strand in the production of progeny virus [75,76].…”

“…The question whether complementary strand synthesis starts on the tail of the rolling circle or whether the synthesis is initiated on the segregated circular viral strand is not completely solved. In vitro studies in the ~X system during stage I replication have shown that once assembled on the viral strand, the primosome which is responsible for the initiation of complementary strand synthesis remains bound after the circle has become a covalently closed, supercoiled duplex [24,35,58,59]. Primosome-associated ~X RFI or ~,X RFIV DNA is a much better substrate for cleavage with gene A protein than superhelical RFI DNA, as shown by the 20-fold enhancement of the initial rate of DNA synthesis [35].…”

“…In vitro studies have examined both pathways. Viral strand synthesis has been studied with extracts and purified proteins without complementary strand synthesis [56], followed by [57] or concomitant with [58,59] complementary strand synthesis. In general, the results obtained are in agreement with the in vivo studies.…”

Conservation of the primosome in successive stages of phi X174 DNA replication.

“…An apparent requirement for localized single-strandedness at the gene A cleavage site (13,14), facilitated by superhelicity, has implicated gyrase in this process (15). Growth of the nascent viral strand ;provides the template for initiation of a complementary strand by primosome action of a ssDNA --RF system identical to that used to initiate synthesis of the parental RF (16).…”