We previously reported our finding that human cells contain glycosylase activity toward all four etheno bases formed in DNA by chloroacetaldehyde and related bi-functional aldehydes. By enzyme purification, including FPLC, we isolated two separate glycosylase activities for 1,N6-ethenoadenine (epsilon A) and for 3,N4-ethenocytosine (epsilon C) respectively, from crude HeLa cell-free extracts, which also contained a number of well-described glycosylases. When Mono-S FPLC purified proteins were assayed against defined oligomers containing either epsilon A or epsilon C, it was found that epsilon A and epsilon C glycosylases were completely separated. It could also be demonstrated that each enzyme bound to and cut only epsilon A- or epsilon C-containing oligomers respectively. There was no overlap in specificity for these two substrates. Several other human glycosylase substrates were also tested and none were cleaved by epsilon C glycosylase. The epsilon C glycosylase activity identified in the present study apparently represents a previously unknown glycosylase. This work also suggests that enzyme recognition of closely related DNA adducts may depend upon subtle changes in local conformation.
Relationships between the concentration of exonuclease, DNA polymerase, and DNA ligase participating in the DNA incision‐excision repair pathway have been derived by applying steady state conditions to concentration of the species of interest. The analysis utilizes concentrations of damage excised, gapped DNA (DNAm—n) and gap filled unligated DNA (DNAm) at the steady state as the parameters to measure the extent of DNA repair. The concentrations of DNAm—n and DNAm have been expressed in terms of experimentally determinable constants such as Km, catalytic constants for the enzymes, enzyme, and substrate DNA concentrations, making it possible to verify the theoretical predictions. The analysis predicts a low ratio of exonuclease to polymerase to observe any significant repair activity, a conclusion consistent with in vitro observation of Hamilton et al. (1974) that significant DNA repair is observed when exonuclease: polymerase ratio is 0.5. The analyis predicts fairly large concentrations of DNA ligase to minimize DNAm concentration at the steady state. Under specific conditions, reversal of ligation has been predicted and has been suggested as a possible mechanism of initiation of multi‐centered DNA replication in eukaryotes. This suggestion has been substantiated by experimental demonstration that DNA nicked in the presence of DNA ligase and AMP is substrate for M. Luteus DNA polymerase but not for T4 DNA polymerase, suggesting that adenylated DNA intermediate is formed and only cleaved by 5′—3′ exonuclease which is present in vivo, and has been isolated and characterized. The relationships have been numerically evaluated for different enzyme concentrations using derived and literature values for the constants. The potential application of these results for in vitro introduction of “errors” into DNA has been indicated.
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