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3.3 DNA replication

(a)

(b)

Linear eukaryote chromosome

Direction of replication

Replication from multiple sites of origin

Origin of replication

Bidirectional replication forks

Direction of replication

Replication forks

Completion of replication

Template DNA

Newly synthesized DNA

Two daughter chromosomes

E. coli chromosome

Partially replicated chromosome

Figure 3.15 DNA replication. (a) Bidirectional replication of the E. coli chromosome. Template strands are blue; newly synthesized strands are red. (b) Multiple bidirectional replication forks in a eukaryotic chromosome. Source : Snape, A. & Papachristodoulou, D. (2018). Biochemistry and Molecular Biology (6th ed.). Oxford University Press.

its attachment to protein structures restrict it from rotating. The resulting supercoils will oppose the separating process, bringing replication to a halt. To prevent this, enzymes called topoisomerases act on the DNA. They transiently break the sugar–phosphate back bone of either one or both strands and allow the supercoils to relax before rejoining the strands with no loss of or change to the base pair sequence. Replication machinery of prokaryotic cells The basic process of DNA replication is similar in prokary otes and eukaryotes, but there are some differences, for exam ple in the enzymes involved. We will look in some detail at prokaryotic replication using the well-studied bacterial species Escherichia coli ( E. coli ) as an example and will review impor tant differences between prokaryotes and eukaryotes later in this section (see Comparison of DNA replication and repair in prokaryotes and eukaryotes). The enzymes that catalyse replication are called DNA polymerases ; they polymerize nucleotides into DNA, using deoxy nucleoside triphosphates ( dNTPs ) as substrates. The reaction is illustrated in Figure 3.17. The incoming deoxynucleoside triphosphate (specifically dATP in Figure 3.17) base pairs with the template strand through hydro gen bonding, which is spontaneous and energetically favourable. The enzyme-catalysed reaction is the formation of a new phospho diester bond between the 3 ′ hydroxyl of the preceding nucleotide in the new DNA chain, and the α-phosphate of the new dNTP. The energy for this reaction is provided by release of inorganic pyro phosphate (PP i ) from the incoming dNTP and its subsequent hydrolysis into two phosphate ions. There are three major DNA polymerases in E. coli , Pol I, II, and III —named in order of their discovery. DNA synthesis at the replication fork is mainly catalysed by Pol III , but Pol I plays an

Topoisomerase enzymes relieve supercoiling ahead of the replication fork DNA strand separation by helicase presents problems due to the introduction of additional supercoiling into the DNA. You can illustrate this problem for yourself using twisted cord or rope as shown in Figure 3.16. The cord is held or clamped at one end and this prevents it from rotating and relaxing as the strands are separated. DNA in the cell is similarly not free to relax ahead of the rep lication fork. In E. coli the closed-circle chromosome effectively ‘clamps’ the DNA, while in eukaryotes the length of the DNA and

Figure 3.16 Supercoiling of DNA. Supercoiling ahead of the replication fork is analogous to the coiling and twisting of a cord as you try to separate the strands. Topoisomerase enzymes cut and rejoin the DNA, allowing the supercoils to relax so that strand separation and replication can continue.

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