All of a cell’s DNA is under constant surveillance for damage, and the repair mechanisms we have described act on all parts of the genome. However, cells have a way of directing DNA repair to the DNA sequences that are most urgently needed. They do this by linking RNA polymerase, the enzyme that transcribes DNA into RNA as the first step in gene expression, to the nucleotide excision repair pathway. As discussed above, this repair system can correct many different types of DNA damage. RNA polymerase stalls at DNA lesions and, through the use of coupling proteins, directs the excision repair machinery to these sites. In bacteria, where genes are relatively short, the stalled RNA polymerase can be dissociated from the DNA; the DNA is repaired, and the gene is transcribed again from the beginning. In eukaryotes, where genes can be enormously long, a more complex reaction is used to “back up” the RNA polymerase, repair the damage, and then restart the polymerase.
The importance of transcription-coupled excision repair is seen in people with Cockayne syndrome, which is caused by a defect in this coupling. These individuals suffer from growth retardation, skeletal abnormalities, progressive neural retardation, and severe sensitivity to sunlight. Most of these problems are thought to arise from RNA polymerase molecules that become permanently stalled at sites of DNA damage that lie in important genes.
The Chemistry of the DNA Bases Facilitates Damage Detection
The DNA double helix seems optimal for repair. As noted above, it contains a backup copy of all genetic information. Equally importantly, the nature of the four bases in DNA makes the distinction between undamaged and damaged bases very clear. For example, every possible deamination event in DNA yields an “unnatural” base, which can be directly recognized and removed by a specific DNA glycosylase. Hypoxanthine, for example, is the simplest purine base capable of pairing specifically with C, but hypoxanthine is the direct deamination product of A (Figure 5–43A). The addition of a second amino group to hypoxanthine
Figure 5–42 The recognition of an unusual nucleotide in DNA by base-flipping. The DNA glycosylase family of enzymes recognizes specific inappropriate bases in the conformation shown. Each of these enzymes cleaves the glycosyl bond that connects a particular recognized base (yellow) to the backbone sugar, removing it from the DNA. (A) Stick model; (B) space-filling model.
Figure 5–43 The deamination of DNA nucleotides. In each case, the oxygen atom that is added in this reaction with water is colored red. (A) The spontaneous deamination products of A and G are recognizable as unnatural when they occur in DNA and thus are readily found and repaired. The deamination of C to U was also illustrated in Figure 5–38; T has no amino group to remove. (B) About 3% of the C nucleotides in vertebrate DNAs are methylated to help in controlling gene expression (discussed in Chapter 7). When these 5-methyl C nucleotides are accidentally deaminated, they form the natural nucleotide T. However, this T will be paired with a G on the opposite strand, forming a mismatched base pair.
produces G, which cannot be formed from A by spontaneous deamination, and whose deamination product (xanthine) is likewise unique.
As discussed in Chapter 6, RNA is thought, on an evolutionary time scale, to have served as the genetic material before DNA, and it seems likely that the genetic code was initially carried in the four nucleotides A, C, G, and U. This raises the question of why the U in RNA was replaced in DNA by T (which is 5-methyl U). We have seen that the spontaneous deamination of C converts it to U, but that this event is rendered relatively harmless by uracil DNA glycosylase. However, if DNA contained U as a natural base, the repair system would not be able to distinguish a deaminated C from a naturally occurring U.