Note that the major excision products from undamaged and T T substrates are nt in length.The photoproduct causes one nucleotide slower migration than expected.The phosphorothioate and methylphosphonate modifications may also cause slightly anomalous migration of the oligomer.This is because both the nucleobases and the phosphodiester bonds are rather reactive and prone to modification by both extrinsic and intrinsic agents.For comparison, all excision reactions were under substratelimiting conditions, and the sources of excision nuclease were cellfree extracts prepared from mammalian cells.With undamaged DNA any combination of incisions about nt apart, bracketing the label, release the appropriate size fragments.Since even undamaged DNA can <a href="http://inhibit09.online/archives/217"></a>
assume the conformation of the preincision complex with low but finite probability, it is not surprising that undamaged DNA is a substrate for excision nucleases.In one of those studies, however, uniformly radiolabeled plasmid DNA was used in a nicking assay that is incapable of detecting an excinuclease mode of action. The second study used linear DNA uniformly labeled with P as a substrate in an excision assay, and ntlong oligomers were released instead of the characteristic ntlong oligomers. It is very likely that certain DNA sequences would be more susceptible to attack by excision nuclease than others.We have tested three different random sequences and found a similar level of excision by the excision nuclease.A more extensive survey, however, is likely to identify certain sequences and conformations with increased susceptibility to excision nuclease.It has been speculated that when RNA polymerase stalls at natural transcriptional pause sites the transcriptioncoupled repair machinery is activated in a manner similar to RNA polymerase stalling at a lesion and that such activation of the transcriptioncoupled repair system leads to gratuitous and potentially mutagenic repair.Currently there is no experimental evidence for gratuitous repair initiated by stalled RNA polymerase.However, there are several reports that show that transcribed DNA is mutated at higher frequency than nontranscribed DNA. Whether this increased mutation frequency is due to transcriptioncoupled gratuitous DNA repair or the increased susceptibility of singlestranded DNA in the transcription bubble to various DNA damaging agents is not known at present.This excision and resynthesis may not be totally innocuous, since it may introduce spontaneous mutations into undamaged DNA as is shown in the following calculation.As is apparent, with the unique substrate and assay system we use, undamaged DNA is excised at a rate of about that of the photoproduct, which is the best natural substrate for the enzyme and is used as the gold standard for other substrates.However, in calculating the susceptibility of undamaged DNA to excision nuclease activity with the photoproduct as a reference, a correction factor must be introduced for the relative abundance of the targets.Essentially all of the excision products from the substrate arise from a single lesion, whereas the excision products from undamaged DNA arise from dual incisions over about a nucleotide region in a variety of combinations that bracket the radiolabel. Hence, in calculating the efficiency of the enzyme on an undamaged nucleotide, a correction factor of is introduced, making the actual efficiency of an undamaged base relative to that of a.