21 DNA Damage and Mutations

At many points in the semester, we’ve brought up mutations to consider their effects on cell structures and functions. Mutations are heritable changes to genetic information—a change to the DNA sequence. Because DNA is replicated and passed from parent cell to daughter cells, a mutation leads to a permanent change in the genetic information that will also be present in the progeny of the cell with the mutation (Figure 21.1). Mutations can arise from errors in DNA replication that are not corrected, or DNA damage that is not correctly repaired prior to DNA replication. Importantly, the error or damage alone is not a mutation, as there is a chance for repair to occur. After DNA replication, however, there is no way for the cell identify the mutation, and the change is now permanent in that cell’s lineage.

While mutations can sometimes have beneficial effects and are necessary for evolutionary change, and many mutations are neutral to cell function, some mutations can be devastating. In the context of cell division, mutations that affect how cells divide can lead to cancer, and we will consider one such mutation in Chapter 22. Because the effects of mutations are unpredictable, cells have many safeguards to prevent mutations, such as exceptionally accurate DNA replication and detection and repair of DNA damage. We shall see that most methods of detecting and accurately repairing genetic information rely on the structure of DNA itself.

Chapter Outline

Section 21.1 Errors in DNA Replication

Section 21.2 DNA Damage

Section 21.3 Small- and Large-Scale Mutations

Section 21.1 Errors in DNA Replication

As discussed in previous chapters, in DNA replication, the entire genome is duplicated so that it can be equally divided among daughter cells. In eukaryotic cells, DNA replication occurs during S phase of the cell cycle and is carried out by DNA polymerase. This process can be quite accurate—in human cells, more than 1 billion base pairs can be replicated with only one error! This extreme level of accuracy is aided by the accuracy of the DNA polymerase enzyme itself and its ability to check its work as it goes (proofreading), along with the ability to fix errors just after replication has occurred (mismatch repair).

One mechanism that helps keep the mutation rate low is proofreading. As DNA polymerase is actively replicating a sequence, the enzyme can sense that a wrong nucleotide has been added. Remember the base pairing rules, A with T and G with C—these pairs form the correct hydrogen bonds for a double helix with uniform width (review Figure 20.3). Any other combination will result in a distortion to the double helix (Figure 21.2a). After DNA polymerase senses the error, it removes the incorrect nucleotide and then resumes the replication process (Figure 21.2b-c).

A second mechanism that reduces the mutation rate is mismatch repair. Occasionally, DNA polymerase does not correct an error as it goes, and a mismatch is created (Figure 21.3a). Once again, the distortion in the double helix is a structural clue that repair needs to occur. However, to accurately repair the error, the cell needs to know which is the incorrect nucleotide. Over time, cells add chemical modifications to DNA strands, but a newly synthesized strand lacks these marks, thus allowing cells to distinguish parent from daughter strands for a brief time in the cell cycle. Assuming that any mismatches are due to errors in replication, enzymes in the cell remove the DNA segment surrounding the mismatch on the new (daughter strand) (Figure 21.3b). Then, DNA polymerase fills in the missing sequence, using base pairing with the parent strand, and DNA ligase seals the gap in the DNA backbone (Figure 21.3c).

As mentioned earlier, DNA replication errors are rare and often corrected before they become mutations. However, the sheer size of the human genome (around 6 billion base pairs of DNA) means that even with a very low error rate, each cell cycle produces daughter cells with a few new mutations. Every cell division is thus an opportunity for DNA replication errors to arise, and cells that divide more often develop more mutations.

Section 21.2 DNA Damage

Another source of mutations is incorrectly repaired DNA damage. Exposure to environmental factors that cause DNA damage (mutagens) are a source of mutations. Additionally, many naturally occurring chemical reactions also damage DNA on a regular basis. Moreover, DNA damage is not restricted to types of cells or certain points in the cell cycle, as is the case with DNA replication errors. Overall, though, you shouldn’t be too alarmed—there are many robust processes in place to detect and mitigate most of this damage when it occurs. And while there are number of different ways in which DNA can be damaged, DNA repair strategies generally follow a simple pattern: recognize that damage has occurred, identify the strand with the damage and remove those sequences, then use the undamaged DNA strand as the template for repair.

You have likely heard warnings about some environmental factors due to their potential to act as mutagens and cause mutations. Ultraviolet radiation from the sun can cause two adjacent thymine bases to form covalent bonds with each other (Figure 21.4a). The resulting distortion in the double helix affects correct base pairing and impedes DNA replication. Specialized enzymes detect these thymine dimers so they can be removed and replaced by the process described above. Additionally, exposure to X-rays cause a different type of damage: breaks to both strands of the sugar-phosphate backbone of DNA (Figure 21.4b). This type of damage is particularly problematic because there is potential for a large amount of genetic information to be lost if the cell divides before the damage is repaired.

While one can try to reduce exposure to mutagens to minimize mutation risk, DNA damage due to compounds produced by metabolism and the normal cellular environment is unavoidable. One very common type of DNA damage is depurination, or loss of a purine (A or G) base, caused by hydrolysis of the base from the sugar. This happens thousands of times per cell per day, leaving a gap in the genetic information (Figure 21.5). Luckily, this damage is unlikely to lead to a mutation because the damaged base is easily recognized, and the other DNA strand contains the information to replace the missing purine.

Another common type of DNA damage is deamination, or removal of an amino group, from a nitrogenous base, most often cytosine. The deamination of cytosine creates uracil, a base typically only found in RNA (Figure 21.6a), and changes a G:C base pair into a G:U base pair. For reasons discussed in Chapter 24, mammalian cells frequently add methyl (-CH3) groups to certain cytosine bases to produce 5-methylcytosine (Figure 21.5b). This does not affect how cytosine base pairs, so gene expression and DNA replication aren’t affected. However, when 5-methylcytosine is deaminated, the base thymine is produced (Figure 21.6b). Thus, the DNA damage changed a G:C base pair to a G:T base pair. For both scenarios in Figure 21.6, consider the criteria for accurate DNA repair. Can the cell recognize that damage has occurred? Can the cell reliably identify the strand with the damage? Is there an undamaged strand for repair? If any of these conditions are not met, there is a higher likelihood that repair will lead to a mutation.

As noted earlier, DNA damage alone does not create a mutation: DNA replication must occur to create the heritable change to the genetic information. Cells constantly surveil the DNA to identify and repair DNA damage, and this is especially important for dividing cells. The DNA damage checkpoint in late G1 (Section 19.2) is another process that reduces the likelihood that mutations will be created. Altogether, the processes in cells are very effective for maintaining the integrity of genetic information.

Section 21.3 Small- and Large-Scale Mutations

While the systems for preventing mutations are robust, mutations will inevitably occur over time. Mutations can happen anywhere in the genome, and can range from small changes to the DNA sequence to large-scale rearrangements of chromosomes. We will consider each of these in turn.

Small-scale mutations, or point mutations, are changes that affect one or few base pairs. These could be substitutions, where one base pair is changed to a different base pair in a sequence, or indels (insertions or deletions), in which a few base pairs are added or deleted from a sequence. The effect of the mutations depends both on where in the genome the mutation occurred and the specific change to the sequence. We will focus first on mutations to protein-coding sequences, since we can more easily predict the effects of these mutations.

Figure 21.7a shows several different DNA sequence changes, each with different consequences for the outcome of gene expression. Assume the original DNA sequence is at the start of the protein-coding sequence, and that the bottom strand is the template strand. Remember that transcription builds an RNA that is complementary to the template strand. Once we have the mRNA, we first find the start codon (5’-AUG-3’), then read each group of three nucleotides (codons) from 5’ to 3’, referring to the codon table to determine the amino acid corresponding each codon in translation (Figure 21.7b). On your own, practice transcribing and translating the original DNA sequence, then use the figure to check your work.

The first three mutation examples in Figure 21.7a are substitution mutations. If a mutation results in a different RNA sequence, but does not change the amino acid sequence as shown in the second example, this is a synonymous mutation. Study the codon table in Figure 21.7b carefully. Often, but not always, the third nucleotide of a codon can be altered without affecting the amino acid sequence. In the third example, a mutation alters the second position of the codon. A change in this position always changes the encoded amino acid, creating a missense mutation. In this case, the sequence that once coded for Gln now codes for Arg. If a mutation changes a coding sequence into a stop codon, this is called a nonsense mutation. Notice that the protein sequence just includes the Met amino acid, as no further translation of the protein occurs.

The last example shows an insertion of one base pair after the sequence encoding the start codon. We can see that the second codon and amino acid are different due to the mutation. Although the third codon is not complete, we can already predict that it will code for Ile or Met, while the original sequence coded for something different (the original codon started with U). Insertions or deletions of base pairs lead to many codons and amino acids being altered if the reading frame is affected. Since the reading frame is groups of three nucleotides, adding or removing three base pairs (or any multiple of three) would alter a few amino acids in translation but would not change the reading frame and therefore amino acids before and after the site of the mutation would not change. However, inserting or deleting any other number of base pairs would cause a frameshift mutation, altering all amino acids at the site of the mutation.

How can we predict the impact of these mutations on protein function? As discussed in Chapter 7, we need to consider the properties of the new amino acid,  compare these to the original amino acid, and use this to predict the impact of the mutation on the tertiary protein structure. A synonymous mutation, which doesn’t change the amino acid coded for, does not alter the primary protein structure, and therefore cannot alter the tertiary protein structure. Thus, we would predict no effect on the tertiary protein structure and no effect on protein function, since structure determines function. In contrast, a missense mutation changes the amino acid and could alter protein structure. In the example in Figure 21.7a, the missense mutation changes Gln to Arg, which is a change to the primary protein structure. But is the tertiary structure likely also altered?

Review the structures Gln and Arg as shown in Figure 21.8, remembering to focus on the side chain that differs across amino acids. These amino acids are both hydrophilic, but one is polar uncharged and the other has full a positive charge, which allows for different interactions. This missense mutation could therefore lead to a change in tertiary protein structure and interactions with other molecules, potentially altering the protein’s function. However, if the mutation had resulted in another polar uncharged amino acid added in place of Gln, such as Asn, we might safely predict the tertiary protein structure and protein function of the mutant protein are likely similar to the original protein.

In contrast, nonsense and frameshift mutations have potential to greatly alter the primary and tertiary protein structure. A nonsense mutation shortens the protein produced in gene expression, and the more sequence that is missing as a result, the more likely it is that the protein will be nonfunctional (or have a different function). Similarly, a frameshift mutation results in a protein with a very different amino acid sequence, likely also creating a nonfunctional or altered protein function, depending on the extent of the alteration.

Some mutations affect entire chromosomes or large regions of chromosomes. Figure 21.9 shows several examples of large-scale mutations (chromosomal abnormalities) that can occur due to faulty cell division or DNA repair. For example, errors in DNA replication can lead to parts of chromosomes being missing or duplicated. Faulty repair of double-stranded DNA breaks can lead to sequences on a chromosome being flipped around or attached to another chromosome. Lastly, errors in the spindle checkpoint can lead to gain or loss chromosomes.

While point mutations often alter the expression or function of one protein, large-scale mutations can impact the expression and function of many proteins. The consequences of both small-scale and large-scale mutations depend on which protein or proteins are altered and how this changes their function. At the organismal level, the effects of mutations and can range from unnoticeable to life altering.