What structural features determine repair enzyme specificity and mechanism in chemically modified DNA?

A crucial question in repair is how do enzymes recognize substrates. In surveying the relevant literature, it becomes evident that there are no rules which can be clearly applied. At this time it appears that uracil glycosylase is the only repair enzyme for which all the known substrates can be rationalized on the basis of chemical structure. When surveying the multiplicity of substrates for m3A-DNA glycosylase, it is difficult, on the basis of present knowledge, to explain why 1,N6-etheno-A (epsilon A) is as good a substrate, if not better, than m3A for which the enzyme is named. There is no apparent unifying chemical structure which is required for recognition. It should also be noted that many studies of the mechanism of m3A-DNA glycosylase only utilized-N-3- and N-7-alkylpurines. On this basis, an electron-deficient purine, and later pyrimidine, was considered to be the recognition signal. Since epsilon A and Hx do not fall in this class, this is one illustration of why exploring new substrates becomes important in elucidating enzyme mechanisms. Ubiquitous enzymes, such as 5'-AP endonucleases, are present in both prokaryotes and eukaryotes. The primary function is the same, i.e., repair of an AP site which occurs through natural processes or from the action of DNA glycosylases. There are, however, completely unrelated substrates such as the exocyclic adducts pBQ-dC and pBQ-dG. pBQ-dC is repaired by both the human HAP1 and E. coli Exo III and Endo IV, while pBQ-dG is only repaired by the E. coli enzymes. Yet, when repair of these adducts occurs, it is by the same unusual pathway which differs from the usual base excision repair mechanism. This finding may ultimately not be as unusual as it now seems. The understanding of substrate recognition by repair enzymes, which can have different repair pathways, is complex. For example, three exocyclic derivatives which each have either the same modification (1,N4-epsilon dA and 3,N4-epsilon dC) or the same base with different modifying groups (3,N4-epsilon dC and 3,N4-pBQ-dC) are repaired by three separate enzymes and two mechanism (Figure 9). Investigators have also reported that two separate enzymes and pathways can be found for simple adducts such as m6G and O4T. It is not clear why, for these adducts, both MGMT and excision repair can be utilized. This could be visualized as a "backup" system and may be more common than now known. We cannot think like an enzyme or vice versa. In the absence of enough necessary information, we can only be descriptive. What information is necessary for further understanding? (1) More detailed structural studies of adducts in defined oligonucleotides would be useful. (2) New substrates should be explored. For example, is the mechanism for PBQ-dC (and pBQ-dG) repair unique? This involves guesswork and intuition. (3) For the adducts mentioned in this Perspective and others, understanding enzyme/substrate recognition will be facilitated by cocrystallography and site-directed mutagenesis. (4) Genetic approaches, such as knockouts or targeted mutations in repair genes, should be expanded in order to focus on the physiological role of a specific enzyme. Above all: structure, structure, structure! Enzymologists, organic chemists, physical chemiste, X-ray crystallographers, and others must combine forces if the fundamental problems addressed here are to be understood.