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3.3 DNA replication

5’

5’ 3’

A G

(a)

3’

Last nucleotide added correct.

CH 3

Template strand

Mut S protein identifies mismatch

Mut S

Synthesis site Direction of synthesis Exonuclease site

A G

CH 3

DNA polymerase

Mut S, Mut L and Mut H protein complex forms

(b)

Mut S

Mut L Mut S

Mut H identifies new strand by lack of methylation

A G

CH 3

Last nucleotide added incorrect.

Section of new strand removed

Methyl-directed mismatch repair corrects errors after replication Despite proofreading, the number of mismatches that slip through into the newly synthesized DNA would still give an unacceptable rate of mutation. The cell can therefore replace incorrect bases even after the newly synthesized DNA has been released from the polymerase. In E. coli this is achieved by methyl-directed mismatch repair , which is illustrated in Figure 3.23. Figure 3.23 illustrates what happens when a mismatch of adenine (A) in the newly synthesized strand with guanine (G) in the tem plate strand escapes proofreading correction, causing a distortion in the DNA double helix. The repair system can recognize the dis tortion, but it must also discriminate so that it removes the A base from the new strand and replaces it, rather than replacing the cor rect G base in the template, as that would perpetuate the mutation. How is this strand discrimination made? It depends on DNA methylation. In E. coli some adenine bases in the DNA sequence are methylated by a specific enzyme. This does not affect DNA structure and function. After DNA replication there is a short period when the template strand is methylated but the newly syn thesized strand has not yet been methylated. This allows the repair system to identify the correct, template sequence. The repair sys tem involves proteins designated Mut S, Mut L, and Mut H, which recognize the distortion in the double helix and remove a stretch of Figure 3.22 Simplified diagram of proofreading by DNA Pol I. (a) The situation if the last addition was correct. (b) The situation if the last addition was not correct. The incorrect nucleotide in (b) is more likely to detach from the template strand and slide to the exonuclease site, where it is removed. The newly synthesized chain will then slide back to the synthesis site, where the correct nucleotide is added. Source : Snape, A. & Papachristodoulou, D. (2018). Biochemistry and Molecular Biology (6th ed.). Oxford University Press.

3’

5’

G

CH 3

DNA polymerase III and ligase replace section

G C

CH 3

newly replicated DNA containing the mismatch. DNA polymerase III synthesizes a replacement, thereby correcting the error. DNA ligase completes the repair by sealing the nick. This system increas es the fidelity of replication so that there is a final error rate of less than or equal to one in 10 10 . Comparison of DNA replication in prokaryotes and eukaryotes The general principles of DNA replication in E. coli also apply to eukaryotes, but there are differences in detail. Many eukaryotic DNA polymerases have been identified (15 in humans), but not Figure 3.23 Mismatch repair in E. coli . The repair system includes three proteins, Mut S, Mut L, and Mut H, which recognize the distortion in the double stranded DNA, identify the new (incorrect) strand because it does not contain a methylated base nearby, and drive removal and replacement of the incorrect sequence. Source : Snape, A. & Papachristodoulou, D. (2018). Biochemistry and Molecular Biology (6th ed.). Oxford University Press.

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