How Does DNA Polymerase Prevent Mutations

Mutations are permanent changes of the nucleotide sequence of a particular organism. They may arise due to the errors of DNA replication or external mutagens. The effect of a mutation can be either beneficial or deleterious to the cell. However, cells undergo various types of mechanisms to prevent mutations. DNA polymerase, which is the enzyme involved in DNA replication, is equipped with several mechanisms to prevent errors during DNA replication. During DNA replication, the mispaired bases are replaced by proofreading. Immediately after DNA replication, the remaining mispaired bases are replaced by strand-directed mismatch repair. In addition, the mutations caused by external factors are repaired by several mechanisms such as excision repair, chemical reversal, and double-strand break repair. If the damage is reversible, the cell is subjected to apoptosis in order to avoid passing the faulty DNA to the offspring.

Key Areas Covered

1. What is a Mutation
      – Definition, Types, Causes
2. How Does DNA Polymerase Prevent Mutations
     – Proofreading, Strand-Directed Mismatch Repair

Key Terms: DNA Polymerase, Strand-Directed Mismatch Repair, Mut Proteins, Mutation, Proofreading

What is a Mutation

A mutation refers to a permanent and a heritable change in the nucleotide sequence of the genome. Mutations may arise due to the errors of DNA replication or external factors known as mutagens. The three forms of mutations are point mutations, frameshift mutations, and chromosomal mutations.

Point Mutations

Point mutations are single nucleotide substitutions. The three types of point mutations are missense, nonsense, and silent mutations. Missense mutation alters a single codon of the gene, altering the amino acid in the polypeptide chain. Though nonsense mutations alter the codon sequence, they do not alter the amino acid sequence. Silent mutations alter a single codon to another codon that represents the same amino acid. Point mutations are caused by errors in the DNA replication and by mutagens. Different types of point mutations are shown in figure 1.

Figure 1: Point Mutations

Frameshift Mutations

Frameshift mutations are insertions or deletions of single or several nucleotides from the genome. Insertions, deletions, and duplications are the three types of frameshift mutations. Insertions are the addition of one or several nucleotides to the sequence while deletions are the removal of several nucleotides from the sequence. Duplications are the repeating of several nucleotides. Frameshift mutations are also caused by errors in the DNA replication and by mutagens.

Chromosomal Mutations

Chromosomal mutations are alterations of segments of chromosomes. The types chromosomal mutations are translocations, gene duplications, intra-chromosomal deletions, inversions, and loss of heterozygosity. Translocations are the interchanges of parts of chromosomes between nonhomologous chromosomes. In gene duplication, multiple copies of a particular allele may appear, increasing the gene dosage. The removals of segments of chromosomes are known as intra-chromosomal deletions. Inversions change the orientation of a chromosome segment. Heterozygosity of a gene can be lost due to the loss of an allele in one chromosome by deletion or genetic recombination. Chromosomal mutations are mainly caused by external mutagens and due to mechanical damages to DNA.

How Does DNA Polymerase Prevent Mutations

DNA polymerase is the enzyme responsible for the addition of nucleotide bases to the growing strand during DNA replication. Since the nucleotide sequence of a genome determines the development and functioning of a particular organism, it is vital to synthesize the exact replica of the existing genome during DNA replication. Generally, DNA polymerase maintains high fidelity during DNA replication, only incorporating single mismatched nucleotide per 10⁹ added nucleotides. Therefore, if a mispairing occurs between nitrogenous bases in addition to the standard complementary base pairs, DNA polymerase add that nucleotide to the growing chain, producing a frequent mutation. The errors of DNA replication are corrected by two mechanisms known as proofreading and strand-directed mismatch repair.

Proofreading

Proofreading refers to an initial mechanism of correcting the mispairing base pairs from the growing DNA strand, and it is carried out by DNA polymerase. DNA polymerase carries out proofreading in two steps. The first proofreading occurs just before the addition of a new nucleotide to the growing chain. The affinity of correct nucleotides for DNA polymerase is many times higher than that of the incorrect nucleotides. However, the enzyme should undergo a conformational change just after the incoming nucleotide binds to the template through hydrogen bonds but, before the covenant binding of the nucleotide to the growing strand by the action of DNA polymerase. The incorrectly-base paired nucleotides are prone to dissociate from the template during the conformational change of the DNA polymerase. Hence, the step allows DNA polymerase to ‘double-check’ the nucleotide before adding it to the growing strand permanently. The proofreading mechanism of DNA polymerase is shown in figure 2.

Figure 2: Proofreading

The second proofreading step is known as exonucleolytic proofreading. It occurs immediately after the incorporation of a mismatched nucleotide to the growing strand in a rare instance. DNA polymerase is incapable of adding the second nucleotide next to the mismatched nucleotide. A separate catalytic site of the DNA polymerase known as 3′ to 5′ proofreading exonuclease digests the mispaired nucleotides from the growing chain.

Strand-Directed Mismatch Repair

Despite proofreading mechanisms, DNA polymerase may still incorporate incorrect nucleotides to the growing strand during DNA replication. The replication errors that have escaped from proofreading are removed by the strand-directed mismatch repair. This system detects distortion potential in the DNA helix that is due to mismatched base pairs. However, the repair system should identify the incorrect base from the existing base prior to replacing the mismatch. Generally, E. coli depends on DNA methylation system to recognize the old DNA strand in the double helix as the newly-synthesized strand may not undergo DNA methylation soon. In E.coli, the A residue of the GATC is methylated. The fidelity of the DNA replication is increased by an additional factor of 10² due to the action of strand-directed mismatch repair system. The DNA mismatch repair pathways in eukaryotes, bacteria, and E. coli are shown in figure 3.

Figure 3: DNA Mismatch Repair in Eukaryotes, Bacteria, and E. coli

In the strand-directed mismatch repair, three complex proteins move through the newly-synthesized DNA strand. The first protein known as MutS detects and binds to the distortions in the DNA double helix. The second protein known as MutL detects and binds to the MutS, attracting the third protein known as MutH that distinguish the unmethylated or the newly-synthesized strand. Upon binding, the MutH cuts the unmethylated DNA strand immediately upstream to the G residue in the GATC sequence. An exonuclease is responsible for the degradation of strand downstream to the mismatch. However, this system degrades regions less than 10 nucleotides that are readily re-synthesized by DNA polymerase 1. The Mut proteins of eukaryotes are homologous to that of E. coli.

Conclusion

Mutations are permanent alterations of the nucleotide sequence of the genome that may arise due to the errors in DNA replication or due to the effect of external mutagens. The errors of DNA replication can be corrected by two mechanisms known as proofreading and strand-directed mismatch repair. Proofreading is carried out by DNA polymerase itself during the DNA synthesis. The strand-directed mismatch repair is carried out by Mut proteins just after the DNA replication. However, these repair mechanisms are involved in the maintenance of the integrity of the genome.

Reference:

1. Alberts, Bruce. “DNA Replication Mechanisms.” Molecular Biology of the Cell. 4th edition., U.S. National Library of Medicine, 1 Jan. 1970, Available here.
2. Brown, Terence A. “Mutation, Repair and Recombination.” Genomes. 2nd edition., U.S. National Library of Medicine, 1 Jan. 1970, Available here.

Image Courtesy:

1. “Different Types of Mutations” By Jonsta247 – This file was derived from:Point mutations-en.png (GFDL) via Commons Wikimedia
2. “DNA polymerase” By I, Madprime (CC BY-SA 3.0) via Commons Wikimedia
3. “DNA mismatch repair” By Kenji Fukui – (CC BY 3.0) via Commons Wikimedia