Mechanisms of DNA ReplicationBiochemistry 201Advanced Molecular BiologyApril 14, 1999Doug BrutlagIn 1971 it was known that DNA polymerases, unlike RNA polymerases, couldnot initiate new chains. In addition, it was known that there were three different DNApolymerases in Escherichia coli with different properties. The most abundant (200molecules per cell) was DNA polymerase I which was not highly processive but whichcould mediate nick translation and strand displacement. DNA polymerase II was muchless processive and, hence, much slower. It appeared to be primarily a repair DNApolymerase, limited to filling in short gaps resulting from repair processes. DNApolymerase II is was induced 10 fold in response to SOS repair induction. Finally, therarer DNA polymerase III (10 molecules per cell), while it could not mediate nicktranslation, could synthesize DNA in a highly processive fashion, and was the onlyenzyme that could keep up with the expect rates of DNA replication fork movement(1000 nt incorporation per second). So how could this mixture of DNA syntheticenzymes work to replicate the bacterial chromosome.How are DNA Chains Initiated?This problem was approached using the DNA from small circular singlestranded phages M13, G4 and ØX174. It was known that the first step in the infection ofthese viral DNAs was the conversion of the viral DNA from a single stranded circularform to a duplex replicative form (RF) and this conversion was mediated by host cellenzymes alone. Hence introducing the phageDNA into bacterial extracts and isolating thefactors required for the conversion to RFwould reveal the mechanism of initiation ofDNA chains.M13 is Primed by RNA PolymeraseBased on in vivo results showing thatM13 SS ––>RF reaction was inhibited byrifampicin and other inhibitors of the hostRNA polymerase, extracts of Escherichia coliwere shown to convert M13 SS to a duplexform in a rifampicin sensitive reaction thatrequired the four ribonucleotidetriphosphates in addition to the fourdeoxynucleotide triphosphates. In vitro, ashort RNA primer was made from several locations on the M13 circle. These primerscan server to initiate DNA synthesis by DNA polymerase III. Evidence favoringpriming of DNA synthesis by RNA in M13 includes:• Sensitivity to several inhibitors of the host cell RNA polymerase includingrifampicin, streptolydigen and actinomycin D• Requirement for all four rNTPs in addition to all four dNTPs.• Two stage conversion of SS to RF, the first of which requires rNTP and isrifampicin sensitive, the second of which is rifampicin resistant and requiresdNTP in addition to ATP.• The discovery of a phosphodiester linkage between RNA and DNA in theresulting DNA product.• Persistence of an RNA segment in the RF in the absence of DNA polymerase IPhages ØX174 and G4 Demonstrate Rifampicin Resistant RNA PrimingWhen DNA from eitherG4 or ØX174 is used in similarextracts, DNA synthesis isalso observed, but in arifampicin resistant reaction.Studies with the G4 templateshowed that it requires onlythree enzymes for its SS ––>RF conversion: single-strandDNA binding protein (SSB),dnaG protein (primase) andDNA polymerase IIIholoenzyme (see below). Thesynthesis also requires thepresence of a specific originfor SS DNA strand synthesis. The site of DNA priming is the same one that is used invivo an it has a very particular structure including three hairpin loops. In the presence ofdnaG protein (primase) a short 29 nucleotide RNA is made complementary to the firsthairpin loop. In the presence of DNA polymerase III, or in vivo, the RNA made by dnaGprotein is much shorter (9-11 bases). The primer always starts at a 5’ CTG 3’ templatewith the first two bases of the primer RNA being 5’AG complementary to the TC.Phage ØX174 priming is much more complicated and appears to be more similarto the priming that occurs at the beginning of Okazaki fragments on the discontinuousstrand of DNA synthesis at a replication fork. Initiation starts at a site known as aprimer assembly site (pas) which bears no sequence similarity to the primase bindingsite in G4 or colE1 plasmid. There are extensive regions of DNA secondary structurehowever.The steps in the formation of the ØX primosome involve:• Coating of the single-stranded ØX174 DNA with Escherichia coli SSB DNAbinding protein• Binding of three proteins (PriA, priB and priC) to the primer assemblysequence.• Formation of a complex of six subunits of dnaB protein coupled with sixsubunits of dnaC protein.• Transfer of the complex of dnaB·dnaC to the priA-B-C complex at the primerassembly site via the dnaT gene product. dnaC dissociates at this step and theresulting complex isknown as thepreprimosome.• Binding ofprimase (dnaG) to thepreprimosomecomplex to form theprimosome.The matureprimosome can thenproceed in an ATPdependent fashion totraverse the DNA. Theprimosome canapparently be drivenby either the dnaB proteinin a 3’-5’ direction or by the priA protein in the 5’-> 3’ direction. Both the dnaB proteinand the priA protein in the primosome can serve as a DNA helicase activities. The priAprotein can also displace SSB from in front of the moving primosome. while dnaBcannot and can only move on naked DNA template. During either of these motions, theprimase activity can synthesize primers 11 ± 1 nucleotides in length at various sitesalong the template in a reaction requiring the four rNTPs. Once these primers areextended by DNA polymerase III, the SSB protein is permanently displaced from thesingle-stranded DNA template. Removal of the RNA primers and ultimate sealing ofthe nicks in the DNA require the combined action of 5’ exonuclease of DNA polymeraseI and DNA ligase.It is believed that similar primosome complexes are present on the lagging strandof DNA synthesis because of the sensitivity of DNA synthesis to disruption bytemperature sensitive mutants in dnaB, dnaC, dnaT and dnaG.Primer sites can be generated by nuclease in some plasmid and viral DNAtemplates.The continued replication of ØX174 RF DNA requires a viral encoded proteingpA (gene product A) which serves to nick the covalently closed circular DNA at aspecific site, becoming covalently attached to the 5’ end of the nicked DNA. The gpAprotein then associates with a cellular DNA helicase (rep) and remains bound to it asrep unwinds the two strands of ØX RF. The 3-prime end of the original nick then servesas a primer terminus for extension by DNA polymerase III. At the