Retroviral integrase, one of only three enzymes encoded by the virus, catalyzes the essential step of inserting a DNA copy of the viral genome into the host during infection. Using the avian sarcoma virus integrase, we demonstrate that the enzyme functions as a tetramer. In presteady-state active site titrations, four integrase protomers were required for a single catalytic turnover. Volumetric determination of integrase-DNA complexes imaged by atomic force microscopy during the initial turnover additionally revealed substrate-induced assembly of a tetramer. These results suggest that tetramer formation may be a requisite step during catalysis with ramifications for antiviral design strategies targeting the structurally homologous human immunodeficiency virus, type 1 (HIV-1) integrase.Integrase catalyzes two consecutive transesterification reactions during its in vivo function (1, 2). In the "processing" reaction, the reverse transcriptase-generated DNA copy of the viral genome is trimmed by the endonucleolytic removal of the 3Ј-dinucleotides from its ends. The two processed 3Ј-ends are then inserted into opposing strands of the host DNA in the "joining" reaction via a concerted cleavage-ligation reaction (3-6). Purified integrase catalyzes both reactions on synthetic oligodeoxynucleotide substrates containing viral DNA end sequences in the presence of either Mn 2ϩ or Mg 2ϩ as a cofactor (7-10). In vitro, integrase also catalyzes the apparent reversal of the joining reaction, the "disintegration" activity, on Y-shaped oligodeoxynucleotide substrates (11) as illustrated in Fig. 1. These Y-shaped substrates resemble products of integrase-catalyzed joining and contain a nick immediately 5Ј of the joining site. The disintegration reaction effectively reverses joining by resealing the nick while concurrently displacing the inserted viral sequence. This reaction is routinely used to assay integrase in vitro (8,(12)(13)(14).Numerous structures of integrase catalytic core-containing fragments determined from a variety of retroviral sources have all been dimeric (15-21). However, the two active sites of the subunits in these structures are outwardly oriented on opposite sides of the crystallographic dimers, too far apart (Ͼ50 Å) to be spanned by the requisite 5-6 bp stagger separating the two sites of concerted integration on the host DNA (20). Although several tetrameric models have been hypothesized based on comparisons with the structure of the homologous bacterial Tn5 transposase (20 -22), the structure for neither a full-length integrase nor an integrase-DNA complex has been solved, and the quaternary structure of the catalytically active integrase enzyme remains unknown.We have previously elucidated some mechanistic aspects of substrate specificity for the processing reaction of the avian sarcoma virus (ASV) 1 integrase by presteady-state kinetics (9, 10); however, we were unable to determine the reaction stoichiometry using a synapsed processing substrate due to substrate-induced aggregation, a problem common to r...

The Escherichia coli MutY adenine glycosylase plays a critical role in repairing mismatches in DNA between adenine and the oxidatively damaged guanine base 8-oxoguanine. Crystallographic studies of the catalytic core domain of MutY show that the scissile adenine is extruded from the DNA helix to be bound in the active site of the enzyme (Guan, Y., Manuel, R. C., Arvai, A. S., Parikh, S. S., Mol, C. D., Miller, J. H., Lloyd, S., and Tainer, J. A. (1998) Nat. Struct. Biol. 5, 1058 -1064). However, the structural and mechanistic bases for the recognition of the 8-oxoguanine remain poorly understood. In experiments using a single-stranded 8-bromoguanine-containing synthetic oligodeoxyribonucleotide alone and in a duplex construct mismatched to an adenine, we observed UV cross-linking between MutY and the 8-bromoguanine probe. We further observed enhanced cross-linking in the single strand experiments, suggesting that neither the duplex context nor the mismatch with adenine is required for recognition of the 8-oxoguanine moiety. Stopped-flow fluorescence studies using 2-aminopurine-containing oligodeoxyribonucleotides further revealed the sequential extrusion of the 8-oxoguanine at 108 s ؊1 followed by the adenine at 16 s ؊1 . A protein isomerization step following base flipping at 1.9 s ؊1 was also observed and is postulated to provide additional stabilization of the extruded adenine thereby facilitating its capture by the active site for excision.The effect of cellular damages by reactive oxygen species in carcinogenesis and aging is well documented (1-3). Even in the absence of external oxidative stress, normal metabolic processes produce oxidative damages to DNA (4 -6) requiring repair. Oxidative damage of DNA bases alters their base pairing properties (5, 7) thereby interfering with replication and transcription (8). A predominant lesion found in DNA exposed to reactive oxygen species is 8-oxoguanine, which is especially deleterious due to its ability to form a stable Hoogsteen base pair with adenine in addition to the canonical Watson-Crick base pair with cytosine (9, 10). The facile by DNA polymerase misincorporation of an adenine across from the 8-oxoguanine (11) results in a mutagenic adenine:8-oxoguanine mismatch, a site where further replication prior to repair would lead to C 3 A or G 3 T transversions. In Escherichia coli, the MutY adenine glycosylase (MutY) 1 plays a critical role in preventing mutations stemming from oxidative damages to DNA by excising the adenine from the adenine:8-oxoguanine mismatch.Like all DNA-nucleotide-modifying enzymes, including DNA methylases, base-excision repair glycosylases, and endonucleases (12-15), MutY faces the 2-fold task of recognizing and accessing chemical moieties on DNA bases hidden within the double helix of duplex DNA. These enzymes have evolved an elegantly simple strategy for exposing their targets by rotating the phosphodiester bonds surrounding the nucleotide, causing the target base to be flipped out of the DNA helix (16 -21).Using the E. coli uracil-D...

Integrase catalyzes insertion of a retroviral genome into the host chromosome. After reverse transcription, integrase binds specifically to the ends of the duplex retroviral DNA, endonucleolytically cleaves two nucleotides from each 3-end (the processing activity), and inserts these ends into the host DNA (the joining activity) in a concerted manner. In first-turnover experiments with synapsed DNA substrates, we observed a novel splicing activity that resembles an integrase joining reaction but uses unprocessed ends. This splicing reaction showed an initial exponential phase (k splicing ‫؍‬ 0.02 s ؊1 ) of product formation and generated products macroscopically indistinguishable from those created by the processing and joining activities, thus bringing into question methods previously used to quantitate these reactions in a time regime where multiple turnovers of the enzyme have occurred. With a presteady-state assay, however, we were able to distinguish between different pathways that led to formation of identical products. Furthermore, the splicing reaction allowed characterization of substrate binding and specificity. Although integrase requires only a 3 hydroxyl with respect to nucleophiles derived from DNA, it specifically favors the cognate sequence CATT as the electrophile. These experimental results support a two-site "switching" model for binding and catalysis of all three integrase activities.After infection, retroviruses create a linear DNA copy of their RNA genome that, through the strand-transfer mechanism of reverse transcriptase, places the U3 region of the LTR sequence at one terminus and the U5 region of the LTR sequence at the other terminus (1). Retroviral replication is dependent on the viral protein integrase catalyzing the recombination of the viral DNA genome into the host genomic DNA. Integrase binds to the two blunt-ended viral LTRs, hydrolyzes the terminal two nucleotides to expose a recessed 3Ј-OH of the conserved CA dinucleotide at each of the ends (the processing activity), and inserts these "processed" ends into the host DNA (the joining activity) at sites separated by a virus-specific stagger of six base pairs for avian sarcoma virus (ASV).1 The location of the insertion is nearly random as there is little sequence specificity for the site of recombination within the host genome (2-4). The processing and joining activities are biochemically similar in that both use a hydroxyl group as the nucleophile in an endonucleolytic cleavage. In the case of the processing activity (3Ј-dinucleotide removal), the enzyme is specific in its choice of electrophile (the cognate CATT), whereas the nucleophile (the processed CA-OH) is specified in the joining reaction (strand transfer). Both the ends-processing and joining activities have been reproduced in vitro with purified recombinant integrase and oligonucleotides whose sequences are derived from the retroviral U3 and U5 LTR sequences (5-7). Detailed examination of the processing activity in vitro has revealed that integrase requires the physiol...

Volume 300, uumbcr 1 RBSlJL7YThe UV absorbann: spectrm~~ of maltose-binding pro&in (MBB) showed a bcuudfhl scc of dilTetwcu: curves when mrk&scrins wxc addcd (Fig., I). The absorbance chnngcs were sacurablc and highly rxpmducible (Fig, 2). Comparison of the umount ot nraltodcxtrin added and of the amount of MBP pww ww cunsistcnt with a one-to-ax stoichiomctr? of binding (Fig. 2).Fur the pwposc of ccmp:wing !b.r' qxtw.ii W;IS canvemient to divide the sptittx into thw trgions: above 280 nm, 280 nm to 265 nm, rind below 265 nm. The most dramatic ditrcrcnces betwwn chc spectra obtained with three substrates, mahosc, maltohcptaosc, and cyclic maltohcptaasc, occurred abow 280 :ltn {Fig. 1). AS discussed bd~\~. the ftxctionxl cht\l\gcs \&ti\po to absolurc absorbance were also grcetcst in this region. It is possible that the movement of individuul chwmophores may bc rcsponsiblc for the nunwrous pcuks and valleys obscivccl. MBF cotltnins S tryptopban residues that are responsible for the m;~joritg of the protein absorbance, particularly abovc 29O nm. BcNen 265 nm and 280 nm, addition of chc three substrates generated essentially identical dif&ereilcc spectra. Maltoheptaose produced a small vertical off'sct but l&c the other maltcdextrine showed an identicz 1 patter11 of hx small peaks at 278 nm. 273 nm. and at 265 nm.