Questions asked by other students, with their answers

Question: what is meant by "complementation group", for example in reference to the complementation groups of Cockayne's syndrome and Xeroderma pigmentosum?

Answer: a complementation group can be thought of as a collection of mutations, all of which are in the same gene. The various complementation groups affecting nucleotide excision repair were distinguished from each other by observing that DNA from cells in one complementation group could cure the mutant phenotype of cells from a different complementation group when transfected into those cells, but could not cure the mutant phenotype of cells from the same complementation group. For a fuller discussion of complementation groups, including partial exceptions to the simple definition I've given here, see any genetics textbook.

Question: Here's a related question. In the lecture notes, where you say "that TTD has 3 complementation groups corresponding to 3 gene products:

what exactly do you mean?

Answer: I apologize that my lecture notes are a bit vague here. The first item in the list above, "A", really means TTD-A, which is a complementation group (gene) specific to the disease, TTD. Unfortunately, as indicated in my lecture notes, this gene has not yet been mapped and sequenced, so we don't know anything about the properties of the protein encoded by the gene. The second and third items in the list, "XPB/TTD" and "XPD/TTD" refer to a subset of mutations within the XPB and XPD genes, respectively, that give rise to the symptoms of TTD. These mutations do not (in general) affect nucleotide excision repair (NER), even though XPB and XPD are required for NER. That's because the XPB and XPD helicases are required for both NER and transcription. Some mutations affect their role in NER without affecting their role in transcription, and other mutations affect their role in transcription without affecting their role in NER. The mutations that affect transcription fall into two classes--those that give rise to TTD and those that give rise to Cockayne's Syndrome (CS). The latter group is discussed in my lecture notes immediately after the discussion of TTD complementation groups referred to in this question.

Question: Is dRPase considered a structure-specific enzyme like DNase IV/Fen-1? Also, would KU-87 and -70 also be considered structure-specific, since they recognize ss-ds DNA junctions?

Answer: These are good questions. I think that dRPase is not considered a structure-specific enzyme, or at least not a purely structure-specific enzyme, because it recognizes a distinct chemical moiety, a 2'-deoxyribose 5'-phosphate group, at the 5' end of a DNA chain. If the end did not have a 5' phosphate, dRPase wouldn't recognize it. KU is certainly a structure-specific DNA binding protein. So far as I'm aware, its ATPase activity is dependent on its interaction with DNA, which is structure-specific, so I think it would be considered a structure-specific enzyme.

Question: What is the difference between single-strand annealing (covered under homologous recombination) and the alternative pathway of non-homologous end joining (NHEJ) using microhomologiess?

Answer: Good question. The difference is the size of the stretch of homology. In single-strand annealing, the stretch of homology is several hundred nucleotides, while in NHEJ using microhomologies, it's only a few (perhaps even just one) nucleotides. Single-strand annealing is mediated by the Rad51 protein, and all processes involving Rad51 require several hundred nucleotides of homology.

Question: What does "MSI" stand for, when describing HNPCC?

Answer: MSI stands for "microsatellite instability", and it refers to the fact that the number of repeats in microsatellites (very short, repeated DNA sequences, such as AGTAGTAGTAGTAGTAGT) is unstable in people with defects in mismatch repair. This microsatellite instability is a combined consequence of DNA polymerase slippage during replication, which leads to the newly synthesized strand containing an incorrect number of microsatellite repeats, and failure of mismatch repair, which--if working--would detect the mismatch (as a small loop) and then correct it by inserting or deleting the appropriate number of repeats in the newly synthesized strand.

Question: Can the replication pathway for base excision repair apply to both pure glycosylase and glycosylase with lyase?

Answer: Yes. Sorry I didn't make this clear in my lectures.

Question: Is the only difference between base excision repair and nucleotide excision repair (aside from their steps) that base excision repair is used to fix problems with single bases while NER is used to correct problems with multiple adjacent bases, such as thymidine dimers? What is the difference between the two in terms of application-- i.e. what does the body use BER for that NER cannot fix?

Answer: NER can be used to fix single-base problems as well as problems involving multiple bases. There are two big differences between NER and BER. First, NER can only recognize lesions that distort the DNA helix. Some lesions (such as U paired with A) do not distort the helix and are never repaired by NER, only by BER. Second, BER is limited by the fact that the number of glycoslyases that can recognize damage and initiate repair is limited. There are many types of damage for which no glycosylase exists. These can be repaired only by NER. You are right in thinking that there are many types of damage that can be repaired both by NER and by BER.

Question: Does methylation occur on newly synthesized strands of DNA in E. coli or on the template strands or on both strands? In your lecture, you mentioned that newly synthesized strands are methylated so that E. coli can distinguish between newly synthesized strands and template strands. Then, you mention that "methylation occurs about 10 mins after the replication fork has passed..." Can you explain this to me again?

Answer: Methylation of newly synthesized strands takes place about 10 minutes after the replication fork has passed. This means that there is a stretch of DNA behind the fork in which only the template strand is methylated; the newly-synthesized strand is not yet methylated. The process of MMR in E. coli can take advantage of this asymmetric methylation to distinguish between the newly synthesized strand and template strand. In this region, the MMR machinery always excises the mismatched base in the newly synthesized (unmethylated) strand and replaces it with a new base coded by the template (methylated) strand.

Question: In transcription-coupled NER, what creates the signal once a damaged area is recognized by RNA polymerase--is it the polymerase alone, or the CSA/CSB/polymerase complex? Also, does the polymerase recruit CSA/CSB after recognition of damage, or are they complexed with the polymerase while transcription occurs?

Answer: The details of the signaling mechanism are not yet understood, but it seems clear that CSA and CSB are required as well as RNA polymerase. My understanding is that the evidence suggests that CSA and CSB are complexed with the polymerase while transcription occurs.

Question: How can synthesis-dependent strand annealing (SDSA) start with a break in a diploid set of chromosomes and then jump to homology searching in sister chromosomes? Is this because this occurs during replication?

Answer: I'm not sure I understand your question. Let me see if this helps. SDSA is suppressed during G1 and early S phase, when no sister chromatid is present. SDSA is employed later in S phase and in G2, when a sister chromatid is present. Sister chromatids are preferred for SDSA, both because the nucleotide sequences of sister chromatids should be identical and because sister chromatids are located very close to each other inside the nucleus, whereas homologous chromosomes may be on opposite sides of the nucleus. Thus it's much easier to find the sister chromatid than the homologous chromosome when searching for homologous DNA.

Question: Are 5'-OH groups involved in any type of DNA repair?

Answer: So far as I'm aware, there aren't any eukaryotic DNA repair processes in which 5'-OH groups are intentionally introduced by enzymes in the repair pathway. However, various types of DNA damage can lead to loss of 5'-phosphate and thus to the presence of 5'-OH groups in the DNA. In these cases, cells use the enzyme, polynucleotide kinase (PNK), to transfer phosphate groups from ATP to 5'-OH groups, in order to create the 5'-phosphate groups that are required for final ligation of broken strands. PNK is known to contribute to double-strand break repair (by NHEJ) and to single-strand break repair. It may contribute to other repair processes as well--wherever a 5'-OH exists.