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3.3 DNA replication

Figure 3.20 stops Pol III dissociating prematurely, especially when it has temporarily to detach from the template for the lagging strand, when synthesis of a new Okazaki fragment begins. We turn now to the question of how the Okazaki fragments are linked up into continuous DNA. In E. coli , when Pol III reaches the RNA primer of the preceding Okazaki fragment, it disengages from the DNA, leaving a nick (a break in the sugar–phosphate backbone of one strand of double-stranded polynucleotide) at the DNA/RNA junction. This is where Pol I comes in. Pol I is an astonishing enzyme with three separate catalytic activities on the same molecule: DNA polymerase, 5 ′→ 3 ′ exonuclease , and 3 ′→ 5 ′ exonuclease . Exonuclease activity refers to removal of nucleotides by hydrolysis from the end of a polynucleotide chain (‘exo’ as the nucleotides are ‘nibbled’ away from the end). The action of Pol I in processing Okazaki fragments is illustrated in Figure 3.21. In summary: ■ Pol I attaches to the nicks between successive Okazaki fragments and uses its DNA polymerase activity to add nucleotides to the 3 ′ -OH of the preceding fragment, moving in the 5 ′ → 3 ′ direction. ■ Since, as Pol I moves, it encounters the RNA primer of the next Okazaki fragment, the nucleotides of this are hydrolysed off by its 5 ′→ 3 ′ exonuclease activity and replaced by the new DNA sequence it is synthesizing. ■ Pol I (unlike Pol III) has low processivity —it does not hold on to the DNA template strand firmly and detaches relatively soon after the RNA has been replaced. It does not have the sliding clamp to hold it on to the DNA. ■ When Pol I detaches a nick is left in the chain, which is healed by DNA ligase. DNA ligase catalyses formation of a phosphodiester bond between the 3 ′ -OH of one DNA fragment and the 5 ′ -phos phate of the next. Figure 3.20 Enzymes and proteins at the replication fork of E. coli . Functions of the enzymes are described in the text. SSB (single-strand binding protein) stops the template strands from reannealing. The loop in the lagging strand solves the problem of how DNA polymerase III can continue to move forward (upwards on the page) but synthesizes DNA in the required 5 ′→ 3 ′ direction on both strands. Source : Snape, A. & Papachristodoulou, D. (2018). Biochemistry and Molecular Biology (6th ed.). Oxford University Press.

5´

3´

DNA molecule being replicated

3´

5´

Okazaki fragments

RNA primers

Leading strand

Lagging strand

3´ 5´

3´ 5´

Figure 3.19 Diagram of a replication fork. The leading strand is synthesized continuously, while the lagging strand is synthesized as a series of short (Okazaki) fragments. Source : Snape, A. & Papachristodoulou, D. (2018). Biochemistry and Molecular Biology (6th ed.). Oxford University Press.

towards the replication fork, and the other synthesizing the lag ging strand away from the replication fork, but both moving in the same overall direction. To allow this the lagging template strand is looped, so that for a short distance it is oriented with the same polarity as the leading-strand template. The replication machinery can therefore proceed in the direction of the fork and synthesize both new strands. The sliding clamp protein complex shown in

Parental DNA being replicated

New primer for Okazaki fragment laid down by primase.

Helicase

Primase

SSB

RNA primers

Pol III

Sliding clamp

Previous Okazaki fragment

DNA polymerase I joins at junction of Okazaki fragments.

Leading strand

Lagging strand

3’ 5’

3’ 5’

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