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3 Information: genes and genomes

A second crucial point to understand is that DNA synthesis always proceeds in the 5 ′→ 3 ′ direction with respect to the grow ing strand, because the new nucleotide can only be added to the 3 ′ hydroxyl of the preceding one, not to its 5 ′ end. Because of the antiparallel arrangement of the two DNA strands in the template molecule, this causes a polarity problem for the simultaneous synthesis of both strands at the replication fork, as illustrated in Figure 3.18. To accommodate this one of the new DNA strands, the leading strand , is made continuously, following the direction of the replication fork as it gradually exposes further stretches of the template strand. The other, lagging strand , is made discontinuous ly as a series of short DNA sequences called Okazaki fragments , named after their discoverers Reiji and Tsuneko Okazaki. Okazaki fragments are typically 1000–2000 bases long in E. coli and 100– 200 bases in eukaryotes. As each new stretch of template strand opens up, a new Okazaki fragment is initiated. The fragments are eventually joined together by the DNA ligase enzyme. Note that DNA ligase does not add any new nucleotides to the DNA; it sim ply catalyses formation of a phosphodiester bond between adjacent Okazaki fragments. The synthesis of leading and lagging strands is illustrated in Figure 3.19. A more detailed view of the replication fork including the enzymes and proteins involved is shown in Figure 3.20. Among the key enzymes shown are helicase to unwind the double helix, attached to which is primase, which synthesizes a single RNA prim er for the leading strand and a series of RNA primers at intervals on the lagging strand. SSB, which has a high affinity for single-stranded DNA, but with no base sequence specificity, binds to the separated DNA strands and prevents them from reannealing prematurely. As indicated, in E. coli there are two connected molecules of Pol III at the replication fork, one synthesizing the leading strand

Primer

P O – O O

3´ Template DNA

5´

CH 2

C

O G

3´

Bond formation

OH

O P O – – O O

O P O –

O P O –

5´

O

O CH 2

T

O A

5´

3´

OH

Deoxyribonucleoside triphosphate (dATP, dGTP, dCTP, dTTP, but, in this illustration, dATP)

DNA polymerase

P O

– O

O

3´

5´

CH O G 2

C

3´

O P O – – O O

O P O –

O P O

OH

– + O

O

5´

Inorganic pyrophosphate H 2 O 2P i

CH 2

T

A

O

5´

3´

OH

essential role in DNA replication (described below), as well as in repair. Less is known of Pol II, but it is believed to be associated with certain types of DNA repair. An important point to appreciate is that DNA polymerases cannot initiate a new DNA chain. They can only elongate (add to) a pre existing polynucleotide strand called a primer . In contrast, new RNA molecules can be initiated ‘from scratch’. Therefore, the process of new DNA synthesis actually starts with the synthesis of a short RNA sequence by the enzyme primase . The RNA primer is synthesized by copying the template DNA, and the primase reaction is like that cata lysed by DNA polymerase, except that the substrates are nucleoside triphosphates (ATP, CTP, GTP, and UTP) instead of dNTPs. Once an RNA primer of 10–20 nucleotides has been made, DNA polymerase takes over and extends the chain. The primers are later removed and replaced by DNA, as described later in this section. Figure 3.17 The elongation reaction catalysed by DNA polymerase. The diagram shows the addition of an adenine deoxynucleotide from dATP to the 3 ′ end of the primer DNA strand. The base selected for addition is determined by the base on the template strand, which would be thymine (T) in this example. Note that the synthesis is in the 5 ′→ 3 ′ direction; the chain is being lengthened in the 5 ′→ 3 ′ direction. The dotted line with arrow shows the attack of the 3 ′ -OH on the α-phosphate. Source : Snape, A. & Papachristodoulou, D. (2018). Biochemistry and Molecular Biology (6th ed.). Oxford University Press.

3´

5´

The replication machinery must move in the direction of the green arrow as the parental DNA strands are unwound.

DNA to be replicated

On the other hand, synthesis of the lagging strand of DNA must proceed in the direction of the orange arrow to maintain the 5´ 3´ direction.

5´

3´

Note: this diagram represents one replication fork of a bidirectional replication ‘bubble’ illustrated in Fig. 3.15.

Newly replicated DNA

3´5´

3´

5´

Figure 3.18 The polarity problem in DNA replication. DNA synthesis always proceeds from 5 ′→ 3 ′ , which means that synthesis of the lagging strand (orange arrow) proceeds away from the direction of movement of the replication machinery (green arrow). Source : Adapted from Snape, A. & Papachristodoulou, D. (2018). Biochemistry and Molecular Biology (6th ed.). Oxford University Press.

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