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3 Information: genes and genomes

Pol I attaches here.

Pol I has low processivity and quickly detaches after replacing the primer with DNA; ligase then seals the nick forming a continous DNA strand.

Direction of Pol I movement

Template strand

3´ 5´

rB rB rB rB rB dB dB dB dB 3´ 5´

dB dB dB

5´

dB 3´ — OH

3´ -end of Okazaki fragment

RNA primer at 5´end of Okazaki fragment. This is replaced by DNA

DNA strand attached to primer RNA

If last base is unpaired it is removed by Pol I and replaced.

Figure 3.21 DNA polymerase I (Pol I) actions in processing Okazaki fragments. As well as replacing RNA primers with DNA, Pol I can remove an unpaired (incorrect) base as part of the proofreading process that increases the accuracy of DNA replication. dB, deoxynucleotide; rB, ribonucleotide. Source : Snape, A. & Papachristodoulou, D. (2018). Biochemistry and Molecular Biology (6th ed.). Oxford University Press.

As well as 5 ′→ 3 ′ exonuclease activity, which allows it to remove RNA primers ahead of new DNA synthesis, Pol I has 3 ′→ 5 ′ exonu clease activity, which is involved in proofreading , described in the next subsection. PAUSE AND THINK DNA polymerase and DNA ligase both catalyse formation of a phospho diester bond in the DNA backbone, but what is the key difference between the two classes of enzyme?

in fact, useful in protein synthesis (as discussed in Chapter 5), but it must be avoided in DNA replication. For the most part, this is achieved by DNA polymerase. Watson–Crick base pairs (A:T and G:C) are almost identical to each other in their size and shape, but ‘illegitimate’ base pairs have a different geometry and there fore do not fit well into the polymerase active site. Once a correct dNTP has entered the active site and base pairs with the template, the polymerase very quickly closes around it and places it in the appropriate position to make a phosphodiester bond. This rapid conformational change does not occur if an incorrect dNTP enters the active site. Thus, DNA polymerase is very selective for addition of the correct nucleotide. Polymerase selectivity gives an error rate of around one in 10 5 , but this is still too high and would cause many mutations to occur. However, mistakes are rapidly corrected by proofreading. To achieve this, E. coli Pol I and Pol III have 3 ′→ 5 ′ exonuclease activ ity, which can remove the last added nucleotide from the growing DNA chain if it is incorrect, and allow it to be replaced by a cor rect nucleotide. Proofreading by Pol III occurs during synthesis of the leading and lagging strands and by Pol I during processing of Okazaki fragments. The mechanism of proofreading has been examined in DNA Pol I. The synthesis site and exonuclease sites are sufficiently close on the enzyme surface for the newly formed end of the DNA chain to slide from one site to another (Figure 3.22). An incorrect base is more likely than a correct one to become detached from its template partner and slide from the synthe sis site into the exonuclease site. The slow rate of conformational change of the polymerase enzyme when an incorrect base enters the synthesis site gives time for this to occur. The incorrect base is removed by exonuclease activity, and the new strand then returns to the synthesis site where the correct nucleotide is added and syn thesis resumes. molecule. DNA ligase links two existing DNA molecules together without addition of a new nucleotide.

Answer: DNA polymerase adds a new nucleotide to the growing DNA

Accuracy of DNA synthesis is improved by proofreading and mismatch repair Accurate replication of its DNA sequence is critical for the survival of any organism. Bacteria such as E. coli replicate their genome extremely rapidly, within 40 minutes, but must achieve accuracy at the same time. A human cell must replicate over six billion base pairs for each cell division and a single mistake could cause a genetic disease. The error rate of DNA replication is less than one in a billion. How is this achieved? Proofreading corrects the new strand as it is made When a deoxynucleotide triphosphate (dNTP) enters the active site of a DNA polymerase, it must base pair with the template base. Despite the emphasis we have previously placed on the specific ity of base pairing, hydrogen bonding between non-Watson–Crick base pairs can sometimes occur. Unusual ‘wobble’ base pairing is,

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