Telomere replication
Because eukaryotic chromosomes are linear, DNA replication comes to the end of a line in eukaryotic chromosomes. DNA–pol can add nucleotides in only one direction. In the leading strand, synthesis continues until the end of the chromosome is reached. However, on the lagging strand there is no place for a primer to be made for the DNA fragment to be copied at the end of the chromosome. This presents a problem for the cell because the ends remain unpaired, and over time these ends get progressively shorter as cells continue to divide.
The ends of the linear chromosomes are known as telomeres, which have repetitive sequences that do not code for a particular gene. As a consequence, it is telomeres that are shortened with each round of DNA replication instead of genes. For example, in humans, a six base-pair sequence, TTAGGG, is repeated 100 to 1000 times. The discovery of the enzyme telomerase (Figure 6.3) helped in the understanding of how chromosome ends are maintained. Telomerase attaches to the end of the chromosome and complementary bases to the RNA template are added on the end of the DNA strand. Once the lagging strand template is sufficiently elongated, DNA–pol can now add nucleotides that are complementary to the ends of the chromosomes. Thus, the ends of the chromosomes are replicated.
Source: Source: Clark, Mary, Jung Choi, and Matthew Douglas. Biology 2E. 2018. Figure 6.3: The ends of linear chromosomes are maintained by the action of the telomerase enzyme.
Telomerase is typically found to be active in germ cells, adult stem cells, and some cancer cells. Telomerase is not active in adult somatic cells. Adult somatic cells that undergo cell division continue to have their telomeres shortened. This essentially means that telomere shortening is associated with aging. In 2010, scientists found that telomerase can reverse some age-related conditions in mice, and this may have potential in regenerative medicine. Telomerase-deficient mice were used in these studies; these mice have tissue atrophy, stem-cell depletion, organ system failure, and impaired tissue injury responses. Telomerase reactivation in these mice caused extension of telomeres, reduced DNA damage, reversed neurodegeneration, and improved functioning of the testes, spleen, and intestines. Thus, telomere reactivation may have potential for treating age-related diseases in humans.
For her discovery of telomerase and its action, Elizabeth Blackburn received the Nobel Prize for Medicine and Physiology in 2009.
DNA repair
DNA replication is a highly accurate process, but mistakes can occasionally occur, such as a DNA–pol inserting a wrong base. Uncorrected mistakes may sometimes lead to serious consequences, such as cancer. Repair mechanisms correct the mistakes. In rare cases, mistakes are not corrected, leading to mutations; in other cases, repair enzymes are themselves mutated or defective.
Most of the mistakes during DNA replication are promptly corrected by the proofreading ability of DNA–pol itself (Figure 6.4a). In proofreading, the DNA–pol reads the newly added base before adding the next one, so a correction can be made. The polymerase checks whether the newly added base has paired correctly with the base in the template strand. If it is the right base, the next nucleotide is added. If an incorrect base has been added, the enzyme makes a cut at the phosphodiester bond and releases the wrong nucleotide. This is performed by the 3′ exonuclease action of DNA–pol. Once the incorrect nucleotide has been removed, it can be replaced by the correct one.
Some errors are not corrected during replication, but are instead corrected after replication is completed; this type of repair is known as mismatch repair (Figure 6.4b). Specific repair enzymes recognize the mispaired nucleotide and excise part of the strand that contains it; the excised region is then resynthesized. If the mismatch remains uncorrected, it may lead to more permanent damage when the mismatched DNA is replicated. How do mismatch repair enzymes recognize which of the two bases is the incorrect one? In E. coli, after replication, the nitrogenous base adenine acquires a methyl group; the parental DNA strand will have methyl groups, whereas the newly synthesized strand lacks them. Thus, DNA–pol is able to remove the wrongly incorporated bases from the newly synthesized, non-methylated strand. In eukaryotes, the mechanism is not very well understood, but it is believed to involve recognition of unsealed nicks in the new strand, as well as a short-term continuing association of some of the replication proteins with the new daughter strand after replication has completed.
Another type of repair mechanism, nucleotide excision repair (Figure 6.4c), is similar to mismatch repair, except that it is used to remove damaged bases rather than mismatched ones. The repair enzymes replace abnormal bases by making a cut on both the 3′ and 5′ ends of the damaged base. The segment of DNA is removed and replaced with the correctly paired nucleotides by the action of DNA–pol. Once the bases are filled in, the remaining gap is sealed with a phosphodiester linkage catalyzed by DNA-ligase. This repair mechanism is often employed when ultraviolet light exposure causes the formation of pyrimidine dimers.
A well-studied example of mistakes not being corrected is seen in people suffering from xeroderma pigmentosa. Affected individuals have skin that is highly sensitive to ultraviolet rays from the sun. When individuals are exposed to ultraviolet light, pyrimidine dimers, especially those of thymine, are formed. The thymine dimers distort the structure of the DNA double helix and this may cause problems during DNA replication. People with xeroderma pigmentosa are not able to repair the damage because of a defect in the nucleotide excision repair enzymes, whereas in normal individuals, the thymine dimers are excised and the defect is corrected. People with xeroderma pigmentosa may have a higher risk of contracting skin cancer than those who don’t have the condition.
Figure 6.4: (a) Proofreading by DNA–pol corrects errors during replication. (b) In mismatch repair, the incorrectly added base is detected after replication. The mismatch repair proteins detect this base and remove it from the newly synthesized strand by nuclease action. The gap is now filled with the correctly paired base. (c) Nucleotide excision repairs thymine dimers. When exposed to ultraviolet light, thymines lying adjacent to each other can form thymine dimers. In normal cells they are excised and replaced.
Errors during DNA replication are not the only reason why mutations arise in DNA. As variations in the nucleotide sequence of a genome, mutations can also occur because of damage to DNA. Such mutations may be of two types: induced or spontaneous. Induced mutations are those that result from an exposure to chemicals, ultraviolet rays, x-rays, or some other environmental agent. Spontaneous mutations occur without any exposure to any environmental agent; they are a result of natural reactions taking place within the body.
The effect of a mutation on protein sequence depends in part on where in the genome it occurs, especially whether it is in a coding or non-coding region. Mutations in the non-coding regulatory sequences of a gene, such as promoters, enhancers, and silencers, can alter levels of gene expression, but are less likely to alter the protein sequence. Mutations within introns and in regions with no known biological function are generally neutral, having no effect on phenotype – though intron mutations could alter the protein product if they affect mRNA splicing.
Mutations that occur in coding regions of the genome (Figure 6.5) are more likely to alter the protein product and can be categorized by their effect on amino acid sequence in point mutations and frameshift mutations.
Point mutations are those mutations that affect a single base pair. The most common nucleotide mutations are substitutions, in which one base is replaced by another. These substitutions can be of two types, either transitions or transversions. Transition substitution refers to a purine or pyrimidine being replaced by a base of the same kind; for example, a purine such as adenine may be replaced by the purine guanine. Transversion substitution refers to a purine being replaced by a pyrimidine, or vice versa; for example, cytosine, a pyrimidine, is replaced by adenine, a purine.
Some point mutations are not expressed; these are known as silent mutations. Silent mutations are usually due to a substitution in the third base of a codon, which often represents the same amino acid as the original codon. Other point mutations can result in the replacement of one amino acid by another, which may alter the function of the protein. Point mutations that generate a stop codon can terminate a protein early.
Some mutations can result in an increased number of copies of the same codon. These are called trinucleotide repeat expansions and result in repeated regions of the same amino acid.
Mutations can also be the result of the addition of a base, known as an insertion, or the removal of a base, known as deletion. If an insertion or deletion results in the alteration of the translational reading frame (a frameshift mutation), the resultant protein is usually nonfunctional.
Sometimes a piece of DNA from one chromosome may get translocated to another chromosome or to another region of the same chromosome; this is also known as translocation.
Figure 6.5: Mutations can lead to changes in the protein sequence encoded by the DNA.
Mutations in repair genes have been known to cause cancer. Many mutated repair genes have been implicated in certain forms of pancreatic cancer, colon cancer, and colorectal cancer. Mutations can affect either somatic cells or germ cells. If many mutations accumulate in a somatic cell, they may lead to problems such as the uncontrolled cell division observed in cancer. If a mutation takes place in germ cells, the mutation will be passed on to the next generation, as in the case of hemophilia and xeroderma pigmentosa.
1. Meiosis is a special type of cell division of germ cells in sexually-reproducing organisms used to produce the gametes, such as sperm or egg cells. It involves two rounds of division that ultimately result in four gamete cells with only one copy of each chromosome (haploid). This process is required to produce egg and sperm cells for sexual reproduction. During reproduction, when the sperm and egg unite to form a single cell, the number of chromosomes is restored in the offspring.
References:
- Fowler, Samantha, et al. Concepts of Biology. OpenStax College, Rice University, 2013. Download for free at: https://openstax.org/details/books/concepts-biology.
- Clark, Mary, Jung Choi, and Matthew Douglas. Biology 2E. 2018. Access for free at https://openstax.org/details/books/biology-2e.