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3 Information: genes and genomes

α does not matter much. Mismatch repair occurs in eukaryotes, but mechanisms other than methylation are used for distinguish ing the old and the new strands. Eukaryotes have proteins equiva lent to Mut S and Mut L (but not Mut H), plus additional proteins involved in the process. Mutations in the genes encoding these proteins are associated with an increased risk of colon cancer, evi dence of the importance of mismatch repair in humans. PAUSE AND THINK Why do you think there is no equivalent to the E. coli Mut H protein in mismatch repair in eukaryotes? Replication of the ends of the linear eukaryotic chromosomes pose a particular problem because of the need for an RNA primer and the fact that DNA can only be synthesized in the 5 ′→ 3 ′ direc tion. This is illustrated in Figure 3.24, which shows that short single-stranded ends remain after replication of the lagging strand is complete. As the shortened sequence will act as a template for the next round of DNA replication, this means that eukaryotic chromosomes would become shorter at each successive round. The problem is solved by the existence of a specialized enzyme, telomerase, which can add multiple short repeated sequences to the ends of linear chromosomes, creating the structures known as telomeres. Telomerase is especially active in actively dividing cells such as embryonic cells, and then becomes less active as the

all are involved in DNA replication. In particular, three DNA pol ymerase alpha (Pol α), DNA polymerase delta (Pol δ), and DNA polymerase epsilon (Pol ε), are involved in replication of nuclear chromosomes, while DNA polymerase gamma (Pol γ) replicates the mitochondrial genome. Other eukaryotic DNA polymerases function in DNA repair and recombination. These complex pro cesses are discussed briefly later in this chapter, but without details of the different polymerase enzymes involved. Unlike in prokaryotes, there is no separate primase enzyme. Instead, DNA polymerase α is a multi-subunit enzyme, part of which has primase activity. Pol α is responsible for initiating repli cation of the leading strand and Okazaki fragments on the lagging strand by synthesizing RNA primers. It then adds about 30 nucleo tides to each primer using its DNA polymerase activity, before handing over to Pol δ and Pol ε. Recent research suggests that Pol δ primarily replicates the lagging strand, while Pol ε replicates the leading strand. Both Pol δ and Pol ε are clamped to the template DNA by a protein complex called proliferating cell nuclear antigen (PCNA), the equivalent of the sliding clamp of E. coli . When eukaryotic chromosomes are replicated the nucleosomes create an added complication at the replication fork. The process by which they are dealt with is not fully understood, but it seems that the parental histones are somehow ‘shared out’ between the daughter DNA molecules. Immediately behind the fork nucleosomes are fully reassembled, incorporating new histones as necessary, so that the rep licated DNA immediately regains the normal chromatin structure. Proofreading and mismatch repair occur in eukaryotic DNA synthesis. Pol δ and Pol ε have 3 ′→ 5 ′ exonuclease activity, but Pol α does not. Since Pol α mainly synthesizes the RNA primers, and only short initial stretches of DNA, the lack of proofreading by Pol

by detecting methylation of guanine bases. Methylation of the template is not used to recognize the correct sequence in eukaryotes.

Answer: The role of Mut H is to recognize the original, template sequence

Figure 3.24 The ‘end replication’ problem of linear chromosomes. Removal of the RNA primer leaves a short section (around 100 base pairs) unreplicated at the 5 ′ end of each new strand. At the next round of replication, the new, shorter strands will be replicated and through the same problem the chromosome will shorten again. (Note that for this illustration a very short chromosome is shown. In reality, a single chromosome would have multiple origins of replication, but the ends of the chromosome would still be truncated as shown here). Source : Snape, A. & Papachristodoulou, D. (2018). Biochemistry and Molecular Biology (6th ed.). Oxford University Press.

Unreplicated section

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Removal of RNA primers

DNA synthesis

Origin of replication

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Chromosome to be replicated

RNA primer of Okazaki fragment

Unreplicated section

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