3.5: Mutations and DNA Repair

Introduction

The information encoded in the DNA sequence of a protein-coding gene determines the sequence of amino acids that will be found in the polypeptide. Because the amino acid sequence affects the folding of the polypeptide and the function of the protein, the DNA sequence of a protein-coding gene must be free of errors. The correct DNA sequence also determines the secondary structure and function of rRNA and tRNA molecules. Therefore, the correct DNA sequence is essential to the viability and function of an organism. A change in the normal DNA sequence is a mutation. Mutations in DNA can be caused by errors in replication, mitosis, meiosis or by exposing DNA to a mutagen, an agent that causes a heritable and permanent change in DNA sequence. This agent can be chemical or physical.

Mutagen Classification

DNA mutations can be classified into two categories: small-scale and large-scale. Small-scale mutations affect only one or two nucleotides in a gene, whereas large-scale mutations can affect large stretches of DNA, or even an entire chromosome.

Small-scale mutations include:

• substitution or point mutations
• insertions
• deletions

Large-scale mutations include:

• duplications
• deletions
• insertions
• inversions
• translocations

Small-Scale Mutations

• Substitution mutation: a mutation that exchanges a single nucleotide for another (Figure \(\PageIndex{1}\)). An exchange of a purine for a purine (A for G; G for A) or a pyrimidine for a pyrimidine (C for T; T for C) is known as a transition mutation. The exchange of a purine for a pyrimidine (C/T for A/G; A/G for C/T) is called a transversion mutation. Transition mutations are more common. Substitution mutations can occur as errors made during DNA replication or can also be caused by chemical mutagens. A subsequent round of DNA replication will result in the mutation becoming a change of a base pair. A substitution mutation that occurs in the coding sequence of a protein-coding gene is often called a point mutation.

• Insertion mutation: an insertion mutation adds one or more nucleotides into a DNA sequence (Figure \(\PageIndex{2}\)). They are usually caused by transposable elements, or errors during DNA replication. Insertions in the coding region of a gene may alter splicing of the mRNA (splice site mutation), or cause a shift in the reading frame (frameshift mutation).
• Deletion mutation: a deletion mutation removes one or more nucleotides from the DNA and can occur during DNA replication (Figure \(\PageIndex{2}\)). Like insertions, these mutations can alter a splice site or the reading frame of the gene.

Point mutations that occur in the coding region of protein-coding genes are more likely to affect the amino acid sequence of the polypeptide. These mutations can be categorized further by their effect on amino acid sequence (Figure \(\PageIndex{3}\)) into the following types:
• Frameshift: a frameshift is caused by either an insertion or deletion mutation. Due to the triplet nature of the codon, a frameshift results from the insertion/deletion of a number of nucleotides that is not divisible by three. The resulting shift can disrupt the reading frame of the coding sequence.
• Missense: a missense mutation changes a nucleotide and results in a different amino acid in the coding sequence
• Nonsense: a nonsense mutation results in a premature stop codon
• Silent: a silent mutation is a mutation that changes the codon within a coding sequence but does not change the amino acid. This is due to the degenerate nature of the genetic code. Silent mutations can also be found in non-coding regions of a gene, such as an intron or in a UTR, but because they are in the non-coding region of the gene, they are often simply called substitution mutations.

A point mutation can be reversed by another point mutation. This type of point mutation is called a reversion. In a "true" reversion, the nucleotide is changed back to its original state and the correct amino restored in the polypeptide sequence. However, reversions can occur elsewhere in the coding sequence. This is known as a second-site reversion and produces a complementary mutation that restores gene function.

Large-Scale Mutations

Large scale mutations involve changes to the structure and composition of chromosomes (Figure \(\PageIndex{4}\)). These changes can have significant effects on the number, location, and activity of the genes found on that chromosome.

• Chromosome Duplication: also known as an amplification. A chromosomal duplication is a repetition of a specific chromosomal segment so that multiple copies of the genes within that segment are present.
• Chromosome Translocation: a mutation that exchanges chromosomal regions between non-homologous (i.e. different) chromosomes. Chromosomal translocations are usually harmless, as they do not result in the gain or loss of genetic information. However, in certain cases, the translocation might result in the fusion of two coding sequences. Such a gene fusion can increase the chances of developing cancer.
• Chromosome Deletion: a mutation in which a chromosomal region is deleted, leading to loss of the genes within that region. Chromosomal deletions can have a significant effect on the phenotype of an organism. In humans, several congenital diseases are associated with chromosomal deletions.
• Chromosome Insertion: a mutation in which a chromosomal region is inserted into another chromosome, leading to a gain of genes. This type of insertion is the result of a translocation event, so the insertion into one chromosome is accompanied by a chromosomal deletion in another chromosome. An alternate type of chromosomal insertion moves a chromosome region within the same chromosome. In both types, because no genetic information is lost, chromosomal insertions are usually harmless.
• Chromosome Inversion: a mutation that reverses the orientation of a chromosomal region. Chromosomal inversions may not have a deleterious effect in an organism as long as gene expression is not affected.

DNA Repair

DNA mutations are unavoidable. While some DNA mutations can be beneficial and underlie the evolution of a species, the majority of mutations may be harmful to an organism and must be corrected. Cells have numerous mechanisms for the correction of mutations. These mechanisms are known collectively as DNA repair.

The simplest mechanism of DNA repair is to prevent the mutation in the first place. Both prokaryotic and eukaryotic DNA polymerases have the ability to detect an incorrect nucleotide during DNA replication and replace it with the correct one. This "proofreading" capacity, however, has its limits. The correction must be done immediately. When a DNA polymerase adds the incorrect nucleotide to the 3' end of the daughter DNA strand, it can remove this nucleotide using its 3' to 5' exonuclease activity. An exonuclease is an enzyme that removes nucleotides from the end of a DNA or RNA strand (Figure \(\PageIndex{5}\)). 3' to 5' exonucleases remove these nucleotides at the 3' end of a nucleic acid, shortening the molecule in the 5' direction. A 5' to 3' exonuclease would remove nucleotides from the 5' end of the nucleic acid. The DNA polymerase, when correcting a DNA replication error, will pause replication, remove the incorrect nucleotide from the 3' end and then replace it with the correct one. All prokaryotic DNA polymerases have this "proofreading" activity, along with the eukaryotic DNA polymerases delta and epsilon. This type of proofreading must be done immediately as the DNA polymerase cannot correct its mistakes once it continues to replicate the DNA.

In addition to proofreading by exonucleases, prokaryotic and eukaryotic cells have additional DNA repair mechanisms. The following are summaries of common repair mechanisms found in human cells (Figure \(\PageIndex{6}\)). These mechanisms are similar to those found in other eukaryotic cells and in prokaryotic cells. Each of these repair mechanisms involve another class of repair enzyme known as an endonuclease. An endonuclease is an enzyme that breaks (or "nicks") the phosphodiester bond within the DNA or RNA strand so that a nucleotide can be removed and replaced.

• Base excision repair: a repair mechanism for the correction of a G-T mismatch in DNA. A DNA glycosylase enzyme removes the T base, leaving the remainder of the nucleotide in the DNA strand. An endonuclease "nicks" the DNA strand by breaking the phosphodiester bond upstream of the mutation. Another endonuclease removes the sugar-phosphate remnant. A DNA polymerase replaces the removed nucleotide with the correct one. A DNA ligase replaces the phosphodiester bond.
• Mismatch repair: a repair mechanism for the removal of other base pair mismatches. Several protein complexes bind the parental DNA strand. One acts as an endonuclease to nick the mutated daughter DNA strand. Another acts as a helicase unwinds the DNA so that an exonuclease can remove nucleotides, creating a "gap" in the daughter strand that is filled in by a DNA polymerase. A DNA ligase replaces the missing phosphodiester bonds.
• Nucleotide excision repair: a repair mechanism for the removal of thymine-thymine dimers. A thymine-thymine dimer (T-T dimer) is a type of DNA damage produced by UV light. The UV light causes a bond to form between adjacent thymine bases (i.e. the dimer). This produces a physical distortion in the shape of the DNA helix that can prevent replication. To remove the dimer, a protein complex binds the dimer region. One of the protein complex subunits acts a helicase to unwind the DNA and give access to other components of the protein complex that will act as endonucleases to cut out the region containing the T-T dimer. This gap is filled in by a DNA polymerase and a DNA ligase replaces the missing phosphodiester bonds.

Glossary

Deletion - a mutation that removes a nucleotide from a DNA sequence; produces a frame-shift

DNA damage - any alteration or modification to the DNA structure, which can lead to mutations. This includes single- or double-strand breaks, base modifications, and cross-linking

Endonuclease - an enzyme that hydrolyses a phosphodiester bond within a DNA or RNA sequence

Exonuclease - an enzyme that removes a nucleotide from the end of a DNA or RNA sequence; can remove nucleotides from either the 5' or 3' end

Frameshift mutation - a mutation caused by the insertion or deletion of nucleotides in a DNA codon; shifts the reading frame of the codons, potentially leading to a completely altered protein product

Insertion - a mutation that adds a nucleotide from a DNA sequence; produces a frame-shift

Inversion - a type of mutation where a section of DNA is reversed end to end; leads to changes in gene function or regulation

Mismatch repair - a process that fixes base-pairing mismatches, often caused by errors during DNA replication; involves recognition of the mismatch and excision of the incorrect base

Missense mutation - a mutation that changes the codon and the resulting amino acid

Mutagen - any physical or chemical agent that causes changes or mutations in the DNA sequence; includes UV radiation and certain chemicals

Mutagenesis - the process by which mutations are induced or occur naturally within an organism's DNA

Mutation - a change in a DNA sequence

Nonsense mutation - a mutation that changes codon to a stop codon

Point mutation - a mutation that involves a change in a single nucleotide base pair in the DNA sequence; can result in silent, missense, or nonsense mutations

Reading frame - the way of dividing a sequence of nucleotides into a set of consecutive, non-overlapping triplets called codons

Silent mutation - a mutation that does not affect the function of the protein; often produces a codon that codes for the same amino acid

Substitution mutation - a mutation where one nucleotide base is replaced by another; potentially alters the protein if the change affects the codon

Thymine dimers (T-T dimers) - a specific type of pyrimidine dimer, where two adjacent thymine bases bond together, distorting the DNA structure; typically formed by UV light exposure

Translocation - a mutation involving the rearrangement of parts between two non-homologous chromosomes; can lead to the fusion of genes or disruption of regulatory sequences

UV Radiation - a form of electromagnetic radiation that can cause DNA damage, including the formation of thymine dimers and other mutations