Structure-function analyses of pdgs have been greatly facilitated through the determination of two co-crystal structures of the protein in complex with CPD- or AP site containing DNA [14
]. Although these structures have revealed amino acid residues that interact with the DNA phosphate backbone in both pre-catalytic and post-catalytic complexes, the charge-charge interactions with non-damaged DNAs has not been structurally determined, but inferred from these structures. However, it is reasonable to extrapolate the identity of key residues from these structures that may serve to promote non-target DNA binding, since the core structure of T4-pdg changes remarkably little between the native enzyme and the structures observed in the co-crystal structures [14
The generalized electrostatic interaction between T4-pdg and duplex DNA has been hypothesized to be responsible for the processive nicking activity on defined substrates both in vitro
and in vivo
]. The discovery of the processive nicking activity of the T4-pdg was made by incubating limiting concentrations of T4-pdg with supercoiled, covalently-closed circular form I DNA that had been UV irradiated such that ~ 30% of the plasmid molecules contained two CPDs in close proximity on complementary strands. While following the incision kinetics of T4-pdg, it was observed that concomitant with the conversion of form I DNA to a nicked form II DNA via
incision at CPD sites, there was a significant and linear accumulation of form III DNA molecules created by a DSB [32
]. Velocity sedimentation analyses of the DNA products through alkaline sucrose gradients revealed that there was a bimodal distribution in the molecular weights of the DNAs, such that plasmid DNAs were either fully incised or contained no single-strand breaks. This result not only demonstrated that the kinetics of the accumulation of form III DNA (while starting form I DNA substrate remains in the reaction) is a surrogate measure of the enzyme’s processivity, but also demonstrated that T4-pdg can incise all CPDs within a DNA domain, where the average distance between CPDs varied from 0.27 to 2.7 kb.
processive nicking activity was shown to occur in cells, using an assay in which the kinetics of complete repair of intracellular plasmids containing 5 and 10 CPDs per molecule was measured [16
]. The experimental data showed all the expected parameters of in vivo
processive nicking activity. Following UV irradiation, fully repaired plasmid DNAs accumulated linearly and with no time lag throughout the time course of repair, a result that strongly suggested that at physiological salt concentrations, T4-pdg initiated repair at all CPD sites within a subset of plasmids prior to reinitiating repair on additional plasmids. The overall conclusion that can be drawn from these data is that it is very likely that in a cellular context, if CPDs are in close proximity in complementary strands, wild-type T4-pdg has a high probability to convert these into DSBs that could prove to be deleterious to the cell.
The use of DNA repair enzymes in increasing cellular resistance to environmental toxicants and radiation-induced damages has potential applications in preventing, and possibly treating, a number of human diseases. To a limited degree, this concept has been reduced to practice in the treatment of XP patients through a topical delivery of wild-type T4-pdg (reviewed in [28
]). In this phase II clinical trial, XP patients showed a reduction of the appearance of new actinic keratoses and basal cell carcinomas through daily administration of the enzyme. These data validated and extended earlier cell-based studies in which delivery of T4-pdg enhanced survival in most XP complementation groups tested (except XPG) [24
]. However, in no case, did the survivals come close to achieving UV resistances that are comparable to normal human cells. The reasons for this are likely to be complex and may include the lack of repair of other UV-induced photoproducts, such as 6–4 photoproducts, ring-fragmented guanines, and DNA-protein crosslinks. Additionally, the coordinated hierarchy of transcription-coupled repair and domain-specific repair are not captured through the initiation of BER of CPDs.
However, extrapolation of the use of wild-type T4-pdg in normal human keratinocytes and in normal human populations may face significant challenges, since all attempts to increase cell survival in normal human cells via introduction of T4-pdg has resulted in increased cytotoxicity rather than increased survival. Since the relative survival curves of most XP and normal human cells expressing wild-type-T4-pdg are quite similar, these data suggest that an activity of wild-type T4-pdg creates cytotoxic intermediates which limit the degree of enhanced survival that can be achieved in an XP cell and reduces cell survival in repair-proficient cells. We hypothesize that this cytotoxic intermediate may be the formation of DSBs by the action of T4-pdg cleaving at sites of tightly clustered CPDs in complementary strands.
Thus, the goal of the current investigation was to create a repertoire of T4-pdg and Cv-pdg mutants that retain their catalytic activities as both a DNA glycosylase and AP lyase, but lose or at least minimize, the capacity to incise clustered AP sites and CPDs. Guided by insights gained from analyses of the co-crystal structures of T4-pdg, we were able to predict residues which may contribute to the electrostatic interactions that do not allow dissociation of the enzyme from a DNA domain. It is this affinity that ultimately leads to DSB formation. Our data show that neutralization of one of several residues in fact yielded enzymes with the desired properties and these include T4-pdg R3K, R22Q, R26Q and R117Q and Cv-pdg R3Q, R22Q, and R119Q. These enzymes maintain appropriate glycosylase and AP lyase activities, but fail to incise two AP sites or CPDs in very close proximity. However, the 10–20-fold reduction in catalytic efficiency of T4-pdg R3K and R26Q and Cv-pdg R3Q and R22Q may diminish their efficacy in enhancing overall repair rates. The immediate goal of our future studies will be to express these mutants in both repair-proficient and repair-deficient cells and assay effects on cell survival.
Finally, these data also may provide insight into the mechanisms of target site location. The original descriptions of the processive nicking activity of T4-pdg were described as a “sliding” mechanism. The implicit assumption of that study was that the enzyme remained in contact with the DNA molecule to which it was originally bound and tracked on that molecule until a dissociation event occurred that released the enzyme into bulk solution [32
]. Such tracking on DNA has recently been shown using single molecule diffusion data, in which eight proteins of varying size and function were shown to utilize a rotation-coupled sliding mechanism [38
However, in the case of T4- or Cv-pdg, such analyses have yet to be performed and it is equally plausible that the processive nicking activity could represent a series of hundreds or thousands of dissociations/re-association events, in which the net reaction is manifest as a processive reaction. In this regard, the current study analyzed DNA substrates in which the clustered sites were either in the same strand (the AP nicking assay) or in both strands (the UV irradiated plasmid assay) in which to detect the formation of the double-stranded break, the two CPDs must be in complementary strands.
Analyses of incision data for both assays using the wild-type pdgs reveal that these enzymes readily make dual incisions per binding event, independent of whether the lesions are in the same or different strands. These data favor a mechanism of frequent microscopic dissociation/re-association events such that lesions in either strand are equally well recognized. In contrast, the majority of the mutant enzymes described herein infrequently re-associate with the same DNA molecule, thus producing a random incision pattern.