Within the base excision repair (BER) pathway, the DNA N-glycosylases are responsible for locating and removing the majority of oxidative base damages. Endonuclease III (Nth), formamidopyrimidine DNA glycosylase (Fpg) and endonuclease VIII (Nei) are members of two glycosylase families: the helix–hairpin–helix (HhH) superfamily and the Fpg/Nei family. The search mechanisms employed by these two families of glycosylases were examined using a single molecule assay to image quantum dot (Qdot)-labeled glycosylases interacting with YOYO-1 stained λ-DNA molecules suspended between 5 µm silica beads. The HhH and Fpg/Nei families were found to have a similar diffusive search mechanism described as a continuum of motion, in keeping with rotational diffusion along the DNA molecule ranging from slow, sub-diffusive to faster, unrestricted diffusion. The search mechanism for an Fpg variant, F111A, lacking a phenylalanine wedge residue no longer displayed slow, sub-diffusive motion compared to wild type, suggesting that Fpg base interrogation may be accomplished by Phe111 insertion.

DNA glycosylases are enzymes that perform the initial steps of base excision repair, the principal repair mechanism that identifies and removes endogenous damages that occur in an organism's DNA. We characterized the motion of single molecules of three bacterial glycosylases that recognize oxidized bases, Fpg, Nei, and Nth, as they scan for damages on tightropes of λ DNA. We find that all three enzymes use a key "wedge residue" to scan for damage because mutation of this residue to an alanine results in faster diffusion. Moreover, all three enzymes bind longer and diffuse more slowly on DNA that contains the damages they recognize and remove. Using a sliding window approach to measure diffusion constants and a simple chemomechanical simulation, we demonstrate that these enzymes diffuse along DNA, pausing momentarily to interrogate random bases, and when a damaged base is recognized, they stop to evert and excise it.DNA repair | search mechanisms O xidative DNA damage is produced endogenously during normal cellular metabolism or exogenously by chemical agents and ionizing radiation (1, 2). Oxidatively induced DNA damage resulting from attack by reactive oxygen species accounts for approximately one-half of all DNA base damages (3). Some oxidative base damages, such as thymine glycol, are blocks to DNA polymerases and thus potentially lethal; however, the majority of oxidative base lesions mispair with noncognate bases and are potentially mutagenic (4). Therefore, damaged bases must be repaired to maintain the cell's genomic integrity. With substantial in vivo steady-state levels of oxidative damages, alkylation damages, and apurinic/apyrimidinic (AP) sites among the nearly six billion normal bases, how DNA repair enzymes locate these damages in the sea of undamaged bases has been the subject of much speculation.The DNA repair mechanism responsible for the removal of the majority of endogenous DNA damages is the base excision repair (BER) pathway (4-6). The critical first step in BER is carried out by a DNA glycosylase that, fueled only by thermal energy, locates a damaged base and cleaves the N-glycosyl bond, thus removing the base lesion from the sugar phosphate backbone. Glycosylases are small monomeric proteins that are found in all living organisms and can be separated into different families based on substrate specificity and structural motifs. In Escherichia coli, there are three glycosylases, Nth, Fpg, and Nei, that directly remove oxidized DNA bases, and all three have an associated lyase activity that cleaves the DNA backbone. These glycosylases are members of two structural families, the helixhairpin-helix (HhH) or Nth superfamily and the helix-two turnshelix (H2TH) or Fpg/Nei family (7-9) (Fig. 1A). Interestingly, the HhH superfamily member, endonuclease III (Nth), and Fpg/ Nei family member, endonuclease VIII (Nei), primarily catalyze the removal of oxidized pyrimidines, such as 5,6-dihydroxy-5, 6-dihydrothymine (Tg), whereas formamidopyrimidine DNA glycosylase (Fpg) primarily removes oxidized purines...

DNA methylation profiling of bladder cancer specific gene promoters has been shown to be a sensitive and specific diagnostic tool for early- and late-stage bladder cancer. We have recently shown that a urine-based assay that combines DNA and protein biomarkers can accurately triage patients undergoing evaluation for bladder cancer. As part of this assay, hypermethylation of Twist1 and Nid2 is performed by conventional methylation-specific PCR (cMSP). To increase throughput and lower cost, we have now developed an approach to determine the methylation at single-base resolution of CpG sites using next generation bisulfite sequencing. In addition to Twist1 and Nid2, methylation of the Vimentin promoter, which has been shown to be associated with epithelial to mesenchymal transition (EMT) in bladder cancer, was also evaluated using this technique. To determine the sensitivity of this approach, amplification was performed on control bisulfite converted genomic DNA that was either enzymatically methylated or chemically unmethylated at CpG sites using non-methylation specific PCR. Twist1, Nid2 and Vimentin PCR products were subsequently used as template for emulsion PCR and sequenced using the Ion Torrent PGM. Methylation status was determined by aligning sequencing reads to unmethylated reference sequences. In this assay, 0.4% methylated DNA could be reliably detected in 10 ng of bisulfite converted DNA, more than doubling the sensitivity of the current assay as well as potentially generating a single-base resolution mapping of additional CpG sites not yet quantified for Twist1, Nid2 and Vimentin. The clinical performance of the Next-Gen bisulfite sequencing assays for Twist1, Nid2 and Vimentin is currently being tested using urine samples from patients with bladder cancer as well as from age matched individuals who present with hematuria but who are found to be cancer-free. The technique we have developed could be used to detect other hypermethylated genes in a variety of bodily fluids where percent methylation may be much lower than in tumor tissue samples. Citation Format: Andrew R. Dunn, Shuqiang Li, Cecilia A. Fernandez, Anthony P. Shuber. Detection of bladder cancer-associated gene methylation using next-gen bisulfite sequencing. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 43. doi:10.1158/1538-7445.AM2013-43

The mechanism of helicase translocation on DNA remains controversial and the translocase activity driving their non-canonical functions such as protein displacement is poorly understood. Here, we used single molecule fluorescence assays to study a prototypical superfamily 1 helicase, Bacillus stearothermophilus PcrA, and discovered a progressive looping of ssDNA that is tightly coupled to PcrA translocation on DNA. Variance analysis of hundreds of looping events by a single protein demonstrated that PcrA translocates on ssDNA in uniform steps of 1 nt, reconciling discrepancies in previous structural and biochemical studies. On the forked DNA, rather than acting on the leading strand to unwind the duplex, PcrA anchored itself to the duplex junction and reeled in the lagging strand using its 3'-5' translocation activity. PcrA maintained the open conformation, not the closed conformation observed in crystallographic analysis, during looping-coupled translcation. This activity could rapidly dismantle a preformed RecA filament even at 1nM PcrA, suggesting that the translocation activity and structure-specific DNA binding are responsible for removal of potentially deleterious recombination intermediates.