PMC

A. Ndk activity from E. coli Ndk protein monitored by the decrease in absorbance at 340 nm. B. Glycosylase activity on uracil substrates from E. coli Ndk protein. Cleavage reactions were performed as described in Material and Methods with 50 μM wild type recombinant E. coli Ndk protein and 0.1 μM DNA substrate. C. Human Ndk activity from NM23 proteins monitored by the decrease in absorbance at 340 nm.

hUNG sliding and hopping is unaffected on double stranded DNA substrates containing intervening neutral methylphosphonate (M) linkages. a) Schematic of the substrates used (S5M, S6M). Methylphosphonate positioning was chosen so that the catalytic excision of uracil by hUNG is unaffected (, ). b) Gel images of the site transfer products derived from S5M in the presence and absence of uracil. c) Determination of P_trans, where the observed site transfer (P_trans^obs, eq 1) is calculated at each time point and linearly extrapolated to zero time to determine the true site transfer value (P_trans). Values at 10 and 15 mM uracil were identical indicating measurements were made within the plateau region depicted in ) Summary of the site transfer properties of hUNG for double stranded methylphosphonate containing DNA substrates compared to the all phosphodiester versions. Data for S5 and S6 were reported previously and are shown for comparison (). Values are equal to the mean ± 1 s.d. of at least 3 trials at 0 mM Uracil and 6 at high uracil (3 each at 10 and 15 mM uracil).

(A) Structural alignment of hTDG (PDB ID 5T2W, green) and hSMUG1 (model, purple) with DNA. Protein α-helices (pipes) and β-strands (planks) and DNA coil ribbons are shown. (B) Combination of the DNA from the 5T2W structure with the solvent-accessible surface of hSMUG1. The uracil base was found to be snugly located in the hSMUG1 active-site pocket. The intercalating loop of hSMUG1 precisely fitted into the appropriate void of DNA.

Two-state model for hUNG sliding on nonspecific DNA. In this model hUNG exists in a highly populated “closed” recognition conformation that makes multiple interactions with the phosphate backbone as observed in crystallographic studies, and also a transient mobile “open” sliding conformation that makes little or no interactions with the phosphate backbone (this work). It is reasonable to view the open state as the aborted transition state preceding DNA dissociation. The open state, which must be present at least 5% of the total bound lifetime (see text), allows for fast movement on the DNA while also allowing time for recognition of uracil bases when they are encountered.