No abstract

The base analog, 2-aminopurine (2AP), was used as a fluorescent reporter of the biochemical steps in the proofreading pathway catalyzed by bacteriophage T4 DNA polymerase. "Mutator" DNA polymerases that are defective in different steps in the exonucleolytic proofreading pathway were studied so that transient changes in fluorescence intensity could be equated with specific reaction steps. The G255S-and D131N-DNA polymerases can hydrolyze DNA, the final step in the proofreading pathway, but the mutator phenotype indicates a defect in one or more steps that prepare the primer-terminus for the cleavage reaction. The hydrolysis-defective D112A/E114A-DNA polymerase was also examined. Fluorescent enzyme-DNA complexes were preformed in the absence of Mg 2؉ , and then rapid mixing, stopped-flow techniques were used to determine the fate of the fluorescent complexes upon the addition of Mg 2؉ . Comparisons of fluorescence intensity changes between the wild type and mutant DNA polymerases were used to model the exonucleolytic proofreading pathway. These studies are consistent with a proofreading pathway in which the protein loop structure that contains residue Gly 255 functions in strand separation and transfer of the primer strand from the polymerase active center to form a preexonuclease complex. Residue Asp 131 acts at a later step in formation of the preexonuclease complex.Many DNA polymerases achieve a remarkably high level of DNA replication fidelity due, in part, to exonucleolytic proofreading activity (Refs. 1 and 2; reviewed in Refs. 3 and 4). The decision to proofread is determined by the different rates for extension of a correct compared with a mismatched primerterminus. Nucleotide incorporation is normally rapid from a correct base pair but slow from a mismatched primer-terminus. The slower extension rate in the case of a mismatched primerterminus provides a window of opportunity to initiate the proofreading pathway.In order to study DNA polymerase proofreading in real time, we have used the fluorescence of the base analog 2-aminopurine (2AP) 1 as a reporter. 2AP fluorescence is quenched when 2AP resides in DNA, but fluorescence is restored when the 2AP nucleotide 2AP 2Ј-deoxyribonucleoside 5Ј-monophosphate (d2APMP) is released from DNA by DNA polymerase proofreading activity (5-7). These studies revealed a rate-limiting step in the initiation of the proofreading pathway (5-7). This step encompasses separation of the primer strand from the template strand and transfer of the primer strand from the polymerase active center to form a proposed preexonuclease complex (6, 7). A rate of about 4 s Ϫ1 is detected for this step for the wild type T4 DNA polymerase with the fluorescence assay, which is consistent with a rate of about 5 s Ϫ1 detected with a rapid quench assay (8).2AP fluorescence is also produced by formation of a specific complex with T4 DNA polymerase in which DNA labeled at the 3Ј-end with 2AP interacts with amino acid residues in the exonuclease active center (5). Fluorescence anisotropy studies indic...

Kaposi's sarcoma-associated herpesvirus encodes a chemokine called vMIP-II that has been shown to be a broad range human chemokine receptor antagonist. Two N-terminal peptides, vMIP-II(1^10) and vMIP-II(1^11)dimer (dimerised through Cys11) were synthesised. Both peptides are shown to bind the CXC chemokine receptor 4 (CXCR4). vMIP-II(1^10) was 1400-fold less potent than the native protein whilst the vMIP-II(1^11)dimer was only 180-fold less potent. In addition, both peptides are CXCR4 antagonists. Through analysis of nonstandard, long mixing time two-dimensional nuclear Overhauser enhancement spectroscopy experiments, 13 C relaxation data and amide chemical shift temperature gradients for the N-terminus of vMIP-II, we show that this region populates a turn-like structure over residues 5^8, both in the presence and absence of the full protein scaffold. This major conformation is likely to be in fast exchange with other conformational states but it has not previously been detected in monomeric chemokine structures. This and other studies [Elisseeva et al. (2000) J. Biol. Chem. 275, 26799^26805] suggest that there may be a link between the structuring of the short N-terminal chemokine peptides and their ability to bind their receptor. ß