Biochemistry - The Chemical Reactions of Living Cells, Volume 3 - D. Metzler 1980

Biochemical Genetics and the Synthesis of Nucleic Acids and Proteins
DNA Replication
Viral DNA Replication

In an effort to find the simplest possible systems for studying DNA Synthesis, many researchers turned to small Introduction/6.html">DNA-containing Viruses such as —X174 and M13. They also examined tailed Bacteriophages, including phages λ, T7, and T4, as well as the colicin E-1 plasmid. The advantage of these systems is that it is easier to model METABOLISM/36.html">DNA Replication in Cell extracts, and furthermore, viral and plasmid DNAs are well characterized genetically. In many cases, replication depends on both viral and host cell genes. For example, Mutations in the dnaB, D, E, F, and G genes result in a loss of The ability to support the growth of phage λ, just as when ts-genes are inactivated. At the same time, phage λ retains the ability to replicate in Bacteria with mutant genes A and C. Many viruses, including T-even phages, contain genes that encode their own specific DNA polymerases and other Proteins required for replication.

a. Replicative Forms

It was established that The First stage of —X and M13 viral replication involves The conversion of the single-stranded closed circular DNA of the infecting virus particle into a circular double-stranded replicative form (RF). These double-stranded circular molecules subsequently undergo replication several times, resulting in A large number of RF circular molecules that serve as templates for the synthesis of numerous single-stranded viral (+)-DNAs, which then form mature viruses. Both stages—the conversion of single-stranded DNA into the replicative form and the duplication of the latter—are currently under intensive investigation [201]. For the first stage to occur in —X and M13 viruses, the host bacterium's genome must contain an active DNA polymerase III Gene (dnaE)¹⁾. Nevertheless, it turned out that if purified DNA polymerase III is introduced into the reaction mixture in vitro, it exhibits no activity. Further attempts to obtain a pure enzyme led to the ISOLATION OF A new dnaE gene product, designated as DNA polymerase III*. This enzyme appears to be a higher-molecular-weight polymer than DNA polymerase III [202]. However, another protein with a Molecular Weight of 77,000, known as copolymerase III*, is also required for replication. A tetramer of copolymerase III* and polymerase III is the most active [202]. The functioning of the system also requires the presence of spermidine and ATP (during the initiation of the polymerization phase, ATP is cleaved to ADP and P_i), as well as all four deoxynucleoside triphosphates.

b. RNA Primers

Researchers studying the replication of M13 and —X viruses were in for another surprise. It turned out that RNA polymerase is required for The formation of RF DNA. This served as one of the many arguments supporting the hypothesis that a short RNA fragment (primer) is necessary to initiate DNA synthesis [Equation (15-3)]. Similar observations were made in studies of colicin E-1 plasmid DNA replication. This process is sensitive to rifampicin, a specific inhibitor of RNA polymerase (Supplement 15-A). Along with deoxynucleoside triphosphates, four ribonucleoside triphosphates are also required for DNA replication [203].

Subsequent research established that RNA fragments are present in circular plasmid DNA and may also be contained in the DNA of viruses and E. coli. In the case of phage —X, this RNA Synthesis is mediated by a specific rifampicin-resistant RNA polymerase with a molecular weight of approximately 64,000, whose synthesis is encoded by the dnaG gene of E. coli. It is possible that this very enzyme is required for normal primary DNA synthesis in E. coli [204]. A special Ribonuclease, RNase H, which specifically cleaves the RNA chain of an RNA–DNA hybrid, may play a role in removing the RNA primer. As a result of RNase H action, a gap is formed in the chain, which can be filled by DNA polymerase [205]. However, the question of which enzyme actually removes the RNA primer remains open. In vitro experiments show that RNase H does not remove the RNA completely. It is possible that the RNA primer is removed by the 5'-exonuclease activity of DNA polymerase I (Fig. 15-30) [205a].

Replication of phage —X also requires the products of the dnaB, C, D, and G genes, an "unwinding protein" (Section D.5.c), and two other "factors".

c. "Unwinding" Proteins

Genetic analysis of phage T4 DNA replication revealed that at least five viral genes are required for viral DNA production in E. coli Cells. One of these, gene 43, encodes the phage T4 DNA polymerase, whereas gene 32 encodes a protein known as the DNA unwinding protein [206]. This protein has a higher affinity for single-stranded DNA than for double-stranded DNA, and it binds to the terminal single-stranded region of the DNA double helix, unwinding it and making the purine and pyrimidine bases of the template strand accessible. Genetic studies have established that this protein is essential for both replication and genetic recombination. An analogous protein induced by phage T7 has been isolated, and another protein of this type was obtained from uninfected E. coli [207, 208]. Eukaryotes also possess DNA-binding proteins [208a]. The true function of these "unwinding" proteins at THE MOLECULAR LEVEL has not yet been definitively established. Therefore, further research on DNA replication must focus on identifying the necessary proteins and developing ways to carry out replication in vitro using systems that replicate the process as it occurs in the living cell.

d. Replication of Viral Double-Stranded RF Molecules

It is known that the initiation of phage —X DNA replication requires the presence of a specific gene A in the phage genome. It was recently demonstrated that this gene determines the synthesis of a protein with a molecular weight of 56,000—a specific endonuclease that nicks the viral strand of the RF form, which is necessary to trigger replication [209]. Apparently, following this Cleavage, the synthesis of a short RNA primer is stimulated. DNA replication proceeds in most cases bidirectionally (Section D.2), but the —X replicative form is probably generated in only one direction via a rolling circle mechanism [210]. According to this mechanism [Equation (15-9)], as the newly formed viral DNA strand is synthesized along the complementary (minus) template strand, the original viral DNA (the plus strand) is displaced as a single-stranded "tail".

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Following this, a complementary strand (perhaps in the form of discrete fragments) is synthesized on the single-stranded "tail". Upon one full revolution of the circle, a viral DNA strand twice the usual length is produced. The replication process is completed by its cleavage with an appropriate endonuclease and the ligation of the complementary strands into a circle by a ligase.