Answer to 1996 DNA Repair Midterm Exam Question

In my lectures, I discussed 4 diseases associated with DNA repair--hereditary non-polyposis colon cancer (HNPCC), which is associated with mismatch repair, and Xeroderma pigmentosum (XP), Cockaynes syndrome (CS) and trichothiodystrophy (TTD), which are associated with nucleotide excision repair (NER). Of these, the first two were the best answers. Although CS and TTD involve defects in proteins required for NER, these proteins are also required for transcription. In CS and TTD the mutations affect primarily transcription rather than repair. The question asked for diseases caused by defects in DNA repair pathways. Unless CS and TTD are also accompanied by symptoms of XP, the defects are in primarily in transcription pathways, not repair pathways.

Steps of mismatch repair (described for E. coli; the strand selection and mismatch excision steps in mammalian cells have not yet been characterized):

1. MutS recognizes the mismatch (or small insertion or deletion)
2. MutL stabilizes the MutS-mismatch complex
3. The MutS/MutL complex activates MutH
4. MutH binds to a nearby methylated A in the parental strand
5. MutH nicks the nascent strand opposite the methylated A
6. The UvrD helicase unwinds the DNA from the nick toward the mismatch
7. A single-strand specific exonuclease hydrolyzes the displaced strand
8. The resulting gap is filled in by a DNA polymerase
9. The resulting nick is sealed by DNA ligase

The human genes which, when mutated, can cause HNPCC are MSH2 and MLH1. These are homologs of MutS and MutL, respectively, and they play the same roles in human mismatch repair as MutS and MutL in E. coli (see above) except that in human cells they appear to function as dimers rather than as monomers (as in E. coli).

Steps of NER in mammalian cells:

1. Recognition of and binding to the damaged region by XPA, aided by RPA.
2. Recruitment of TFIIH, with 6 subunits including the helicases XPB and XPD
3. Unwinding of the DNA at and near the damaged region by XPB and XPD
4. Binding of XPF/ERCC1 and XPG (structure-specific nucleases)
5. Double incision of the damaged strand by the structure-specific nucleases, on both the 5' and 3' sides of the damaged site
6. Removal of the damage-containing oligonucleotide (mechanism not clear in mammalian cells; possible participation by the XPB and XPD helicases)
7. Filling in of the resulting gap by a DNA polymerase
8. Sealing the resulting nick with a DNA ligase

It would have been satisfactory to provide a detailed description of the actions of any of the 7 proteins encoded by XP genes. These are summarized here:

• XPA is primarily responsible for recognizing and binding to damaged DNA. In addition, XPA interacts with RPA, TFIIH and ERCC1.
• XPB and XPD are components of TFIIH. XPB is a 3' to 5' helicase, and XPD is a 5' to 3' helicase. They are probably required for initial unwinding to create sufficient single-stranded DNA near the damage to permit binding of XPF/ERCC1 and XPG. They may also be required for subsequent removal of the damage-containing oligonucleotide.
• XPC is a single-stranded DNA binding protein. Its precise role is not yet known, but it may be involved in repair of the non-transcribed DNA strand.
• XPE also plays an as yet uncertain role, possibly in enhancing binding of other proteins to damaged DNA.
• XPF and XPG are involved in recognition of junctions between single- and double-stranded DNA and in cutting at such junctions. They have opposite polarities, making them suitable for introducing nicks in the damaged strand at opposite ends of an unwound (presumably by XPB and XPD) region containing the damaged strand.

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