Mismatched strand identification: When a mismatch occurs, the Mut proteins that identify the mispaired nucleotide(s) must be able to discriminate between the correct strand and the strand with the mismatch. In prokaryotes, discrimination is based on the degree of methylation. GATC sequences, which are found once every thousand nucleotides, are methylated on the adenine (A) residue by DNA adenine methylase (DAM). This methylation is not done immediately after synthesis, so the DNA is hemimethylated (that is, the parental strand is methylated, but the daughter strand is not). The methylated parental strand is assumed to be correct, and it is the daughter strand that gets repaired. [Note: The exact mechanism by which the daughter strand is identified in eukaryotes is not yet known, but likely involves recognition of nicks in the newly synthesized strand.] 2.
Repair procedure: When the strand containing the mismatch is identified, an endonuclease nicks the strand, and the mismatched nucleotide(s) is/are removed by an exonuclease. Additional nucleotides at the 5′-and 3′-ends of the mismatch are also removed. The gap left by removal of the nucleotides is filled, using the sister strand as a template, by a DNA pol, typically DNA pol III. The 3′-hydroxyl of the newly synthesized DNA is joined to the 5′-phosphate of the remaining stretch of the original DNA strand by DNA ligase.
Mutation to the proteins involved in MMR in humans is associated with hereditary nonpolyposis colorectal cancer (HNPCC), also known as Lynch syndrome. Although HNPCC confers an increased risk for developing colon cancer (as well as other cancers), only about 5% of all colon cancer is the result of mutations in MMR.
B. Nucleotide excision repair
Exposure of a cell to UV radiation can result in the covalent joining of two adjacent pyrimidines (usually thymines), producing a dimer. These intrastrand cross-links prevent DNA pol from replicating the DNA strand beyond the site of dimer formation. Thymine dimers are excised in bacteria by UvrABC proteins in a process known as nucleotide excision repair (NER), as illustrated in Figure 30.29. A related pathway is present in humans (see 2. below). [Note: Transcription-coupled repair, a type of NER, fixes DNA lesions encountered during RNA synthesis.] 1.
Recognition and excision of UV-induced dimers: A UV-specific endonuclease (called uvrABC excinuclease) recognizes the bulky dimer and cleaves the damaged strand on both the 5′-side and 3′-side of the lesion. A short oligonucleotide containing the dimer is excised, leaving a gap in the DNA strand. This gap is filled in using a DNA pol I and DNA ligase. NER occurs throughout the cell cycle.
2.
UV radiation and cancer: Pyrimidine dimers can be formed in the skin cells of humans exposed to UV radiation in unfiltered sunlight. In the rare genetic disease xeroderma pigmentosum (XP), the cells cannot repair the damaged DNA, resulting in extensive accumulation of mutations and, consequently, early and numerous skin cancers (Fig. 30.30). XP can be caused by defects in any of the several genes that code for the XP proteins required for NER of UV damage in humans.
C. Base excision repair