Distinguishing Specific and Nonspecific Complexes of Alkyladenine DNA Glycosylase.

Human alkyladenine DNA glycosylase (AAG) recognizes many alkylated and deaminated purine lesions and excises them to initiate the base excision DNA repair pathway. AAG employs facilitated diffusion to rapidly scan nonspecific sites and locate rare sites of damage. Nonspecific DNA binding interactions are critical to the efficiency of this search for damage, but little is known about the binding footprint or the affinity of AAG for nonspecific sites. We used biochemical and biophysical approaches to characterize the binding of AAG to both undamaged and damaged DNA. Although fluorescence anisotropy is routinely used to study DNA binding, we found unexpected complexities in the data for binding of AAG to DNA. Systematic comparison of different fluorescent labels and different lengths of DNA allowed binding models to be distinguished and demonstrated that AAG can bind with high affinity and high density to nonspecific DNA. Fluorescein-labeled DNA gave the most complex behavior but also showed the greatest potential to distinguish specific and nonspecific binding modes. We suggest a unified model that is expected to apply to many DNA binding proteins that exhibit affinity for nonspecific DNA. Although AAG strongly prefers to excise lesions from duplex DNA, nonspecific binding is comparable for single- and double-stranded nonspecific sites. The electrostatically driven binding of AAG to small DNA sites (∼5 nucleotides of single-stranded and ∼6 base pairs of duplex) facilitates the search for DNA damage in chromosomal DNA, which is bound by nucleosomes and other proteins.

Kinetic mechanism for the flipping and excision of 1,N(6)-ethenoadenine by AlkA.

Escherichia coli 3-methyladenine DNA glycosylase II (AlkA), an adaptive response glycosylase with a broad substrate range, initiates base excision repair by flipping a lesion out of the DNA duplex and hydrolyzing the N-glycosidic bond. We used transient and steady state kinetics to determine the minimal mechanism for recognition and excision of 1,N(6)-ethenoadenine (εA) by AlkA. The natural fluorescence of this endogenously produced lesion allowed us to directly monitor the nucleotide flipping step. We found that AlkA rapidly and reversibly binds and flips out εA prior to N-glycosidic bond hydrolysis, which is the rate-limiting step of the reaction. The binding affinity of AlkA for the εA-DNA lesion is only 40-fold tighter than for a nonspecific site and 20-fold weaker than for the abasic DNA site. The mechanism of AlkA-catalyzed excision of εA was compared to that of the human alkyladenine DNA glycosylase (AAG), an independently evolved glycosylase that recognizes many of the same substrates. AlkA and AAG both catalyze N-glycosidic bond hydrolysis to release εA, and their overall rates of reaction are within 2-fold of each other. Nevertheless, we find dramatic differences in the kinetics and thermodynamics for binding to εA-DNA. AlkA catalyzes nucleotide flipping an order of magnitude faster than AAG; however, the equilibrium for flipping is almost 3 orders of magnitude more favorable for AAG than for AlkA. These results illustrate how enzymes that perform the same chemistry can use different substrate recognition strategies to effectively repair DNA damage.

Granulocyte contamination dramatically inhibits spot formation in AIDS virus-specific ELISpot assays: analysis and strategies to ameliorate.

The interferon-gamma (IFNgamma) ELISpot assay has become the most critical tool for HIV vaccine evaluation. External factors affecting ELISpot results must be minimized for the data to be reliably used in vaccine research and development processes. In pre-clinical pigtail macaque studies analyzing HIV/SIV vaccine studies, we detected a strong correlation between levels of granulocytes contaminating PBMC preparations and reduction in the quality and quantity of spots in the IFNgamma ELISpot assay. Acute SHIV infection of macaques worsened granulocyte contamination of PBMC fractions and made the assay much less reliable in detecting SIV-specific T cell immunity compared to intracellular cytokine staining (ICS). This problem could be ameliorated by using an F(ab)(2) form of the MD-1 IFNgamma capture antibody, presumably reflecting that activation of granulocytes in the well by the Fc portion of the standard capture antibody disrupts spot formation. Improving the standard ELISpot assay by using an F(ab)(2) capture antibody should make it more reliable for use in critical vaccine development studies.

Use of the Anderson Sling suspension system for recovery of horses from general anesthesia.

OBJECTIVE: To describe a sling recovery system (Anderson Sling) for horses and to evaluate outcome of high-risk horses recovered from general anesthesia by a sling. STUDY DESIGN: Retrospective study. SAMPLE POPULATION: Horses (n=24) recovered from general anesthesia. METHODS: Complete medical and anesthetic records (1996-2003) for horses recovered from general anesthesia using the Anderson Sling system were evaluated retrospectively. Information retrieved included anesthetic protocol, surgical procedure, recovery protocol, recovery time, and quality of the recovery. Horses were recovered from anesthesia supported by the Anderson Sling in a standing position within a traditional padded equine recovery stall. RESULTS: Twenty-four horses had 32 assisted recoveries; 31 events were successful. No complications associated with the sling or recovery system protocol occurred. One horse was intolerant of the sling's support and was reanesthetized and recovered successfully using head and tail ropes. CONCLUSION: The Anderson Sling recovery system is an effective and safe way to recover horses that are at increased risk for injury associated with adverse events during recovery from general anesthesia. CLINICAL RELEVANCE: The Anderson Sling system should be considered for assisted recovery of equine patients from general anesthesia.