DNA replication in procaryotes

You may recall from Chapter 4 that bacteria multiply by a process of binary fission; before this occurs, each cell must duplicate its genetic information so that each daughter cell has a copy.

DNA replication involves the action of a number of specialised enzymes:

• DNA topoisomerases

• DNA polymerase I

• DNA polymerase III

Replication begins at a specific sequence called an origin of replication. The two strands of DNA are caused to separate by helicases (Figure 11.2), while single-stranded DNA binding proteins (SSB) prevents them rejoining. Opening out part of the double helix causes increased tension (supercoiling) elsewhere in the molecule, which is relieved by the enzyme DNA topoiosomerase (sometimes known as DNA gyrase). As the 'zipper' moves along, and more single stranded DNA is exposed, DNA polymerase III adds new nucleotides to form a complementary second strand, according to the rules of base-pairing. DNA polymerases are not capable of initiating the synthesis of an entirely new strand, but can only extend an existing one. This is because they require a free 3'-OH group onto which to attach new nucleotides. Thus, DNA polymerase III can only work in the 5'-3' direction. A form of RNA polymerase called DNA primase synthesises a short single strand of RNA, which can be used as a primer by the DNA polymerase III. (Figure 11.2).

When replication occurs, complementary nucleotides are added to one of the strands (the leading strand) in a continuous fashion (Figure 11.2). The other strand (the lagging strand), however, runs in the opposite polarity; so how is a complementary sequence synthesised here? The answer is that DNA polymerase III allows a little unwinding to take place and then, starting at the fork, works back over from a new primer, in the 5'-3' direction. Thus, the second strand is synthesised discontinuously, in short bursts, about 1000-2000 nucleotides at a time. These short stretches of DNA are called Okazaki fragments, after their discoverers.

On the lagging strand, a new RNA primer is needed at the start of every Okazaki fragment. These short sequences of RNA are later removed by DNA polymerase I, which

DNA replication takes place at a replication fork, a Y-shaped structure formed by the separating strands. The fork moves along the DNA as replication proceeds.

A primer is a short sequence of single-stranded DNA or RNA required by DNA poly-merase as a starting point for chain extension.

lagging strand lagging strand

leading strand

Figure 11.2 DNA replication takes place at a replication fork. Strands of DNA are separated and unwound by helicase and DNA gyrase, and prevented from rejoining by the attachment of single-strand binding proteins. Starting from an RNA primer, DNA poly-merase III adds the complementary nucleotides to form a second strand. On the leading strand, a single primer is set down (not shown), and replication proceeds uninterrupted in the same direction as the fork. Because DNA polymerase III only works in the 5' ^ 3' direction, on the lagging strand a new primer must be added periodically as the strands open up. Replication here is thus discontinuous, as a series of Okazaki fragments (see the text). From Bolsover, SR, Hyams, JS, Jones, S, Shepherd, EA & White, HA: From Genes to Cells, John Wiley & Sons, 1997. Reproduced by permission of the publishers leading strand

Figure 11.2 DNA replication takes place at a replication fork. Strands of DNA are separated and unwound by helicase and DNA gyrase, and prevented from rejoining by the attachment of single-strand binding proteins. Starting from an RNA primer, DNA poly-merase III adds the complementary nucleotides to form a second strand. On the leading strand, a single primer is set down (not shown), and replication proceeds uninterrupted in the same direction as the fork. Because DNA polymerase III only works in the 5' ^ 3' direction, on the lagging strand a new primer must be added periodically as the strands open up. Replication here is thus discontinuous, as a series of Okazaki fragments (see the text). From Bolsover, SR, Hyams, JS, Jones, S, Shepherd, EA & White, HA: From Genes to Cells, John Wiley & Sons, 1997. Reproduced by permission of the publishers then replaces them with DNA nucleotides. Finally, the fragments are joined together by the action of DNA ligase. Replication is bidirectional (Figure 11.3), with two forks moving in opposite directions; when they meet, the whole chromosome is copied and replication is complete*.

DNA ligase repairs breaks in DNA by reestablishing phosphodi-ester bonds in the sugar-phosphate backbone.

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