Introduction to Molecular Biology: From Cells to Atoms - Anthony Rees, Michael Sternberg 2002

Nucleic Acids and Genes
Replication in Prokaryotes

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Fig. 21.1. Replication in E. coli.

E. coli is a bacterium containing a double-stranded circular DNA. The replication of E. coli DNA has been studied in great detail, and the mechanisms of many stages of this process are well understood. These mechanisms are characteristic of the replication of all prokaryotes. Replication proceeds similarly in eukaryotes, although different Enzymes are involved.

DNA polymerases are enzymes that participate in DNA Synthesis. They add a nucleotide to the —OH group at the 3' end of one of the DNA strands, which therefore grows in the 5' —> 3' direction. Because of this, these enzymes are said to have 5' —> 3' polymerase activity. The synthesis of a new DNA strand requires: 1) a DNA template, which can be in either single- or double-stranded form; 2) deoxynucleoside triphosphates (ATP, CTP, GTP, and TTP); and 3) the 3'-OH group of a primer nucleic acid to which the next base is added. The reaction scheme is shown in Fig. 21.2.

Three DNA polymerases have been isolated — I, II, and III, designated as polI, polII, and polIII. Although polI was the first to be isolated (by Kornberg in 1958), polIII is the principal enzyme catalyzing the Synthesis of the growing DNA strand. PolI is involved in the maturation process (see below), while The Role of polII remains unclear. In addition to their 5' —> 3' polymerase activity, all three enzymes exhibit The ability to degrade DNA by removing NUCLEOTIDES in the 3' —> 5' direction, meaning they act as 3' —> 5' exonucleases. PolI and polIII also possess 5' —> 3' exonuclease activity.

Fig. 21.2.

A Replication fork is the region of the DNA molecule where the synthesis of new DNA is currently taking place. Here, the parental DNA is unwound and exists in a single-stranded form. Each strand serves as a template for the synthesis of new DNA. As synthesis proceeds, the replication fork moves along the molecule, unwinding new segments of the parental DNA until it reaches the termination site. Because DNA polymerases catalyze replication exclusively in the 5' —> 3' direction, and the parental DNA strands are antiparallel, only one of the new strands is synthesized continuously; this is called the leading strand. The second strand, known as the lagging strand, is synthesized as fragments that are subsequently joined (ligated) to form a continuous secondary strand. This process is called maturation. The DNA fragments in question are called Okazaki fragments, named after the researcher who first discovered them. In prokaryotes, they are about 1,000 nucleotides long, whereas in eukaryotes, they are 100–200 nucleotides long.

DNA synthesis requires An RNA primer. Since DNA polymerase requires a free 3'-OH group to function, the question arises as to how DNA synthesis is initiated. Numerous studies have shown that synthesis begins on a small RNA fragment that acts as a primer. First, the enzyme RNA polymerase, which does not require a free 3'-OH group for initiation, synthesizes a short RNA molecule (10–60 nucleotides long) using the parental double-stranded DNA AS A template. This RNA primer remains base-paired with the unwound DNA and serves as the initiation site for synthesis, providing its free 3'-OH group for the attachment of the first DNA nucleotide; this latter step is performed by DNA polymerase III. A similar primer is formed prior to the synthesis of the lagging strand, but since single-stranded DNA serves as the template for its formation, Bacteria often use a different enzyme, primase, to synthesize the RNA primer. The RNA template is removed from the DNA molecule during maturation with the help of polI.

During maturation, the RNA primer is removed from both the 5' end of the leading strand and the 5' ends of the Okazaki fragments, and these fragments are joined together. The removal of the RNA primer is carried out by DNA polymerase I, acting as a 3' —> 5' exonuclease. This same enzyme then replaces the removed RNA with deoxynucleotides using its 5' —> 3' polymerase activity. Finally, the enzyme DNA ligase joins the DNA fragments in the correct order by catalyzing The formation of a phosphodiester bond.

“Proofreading” refers to the removal of incorrect (non-complementary) bases incorporated into the newly synthesized DNA. This process ensures an extremely high fidelity of replication, corresponding to only one error per 10⁹ Base Pairs. Proofreading takes place when an “incorrect” nucleotide is attached to the 3' end of the growing strand, failing to form the proper Hydrogen Bonds with the template. When polIII mistakenly incorporates an incorrect base, its 3'—5' exonuclease activity is triggered, the base is immediately removed, and polymerase activity is then restored. This simple mechanism works because polIII is able to function as a polymerase only on a perfect DNA double helix with completely accurate base pairing. Unwinding of The Double Helix is necessary so that the DNA strands can separate and serve as templates during replication. E. coli possesses a specialized enzyme, known as the rep protein, which performs the unwinding of the double helix. The energy required for this process is derived from the Hydrolysis of ATP. The single-stranded DNA regions generated upon unwinding are stabilized in this state by a variety of Proteins, including single-stranded DNA-binding protein, double-helix destabilizing protein, DNA-binding protein, and double-helix unwinding protein.

Topoisomerases are enzymes that catalyze transitions in DNA molecules associated with Changes in the degree of supercoiling. DNA molecules that differ only in their degree of supercoiling are called topological isomers or topoisomers, hence the name topoisomerases. Some topoisomerases relax supercoiled DNA, whereas others promote the introduction of supercoils (Chapter 26). E. coli DNA gyrase converts circular double-stranded DNA into a negatively supercoiled state. This is essential for relieving positive supercoils that arise during replication (and METABOLISM/31.html">Transcription; Chapter 22) as a consequence of the unwinding of the DNA double helix.