Presteady-state Analysis of Avian Sarcoma Virus Integrase

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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...

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Functional Oligomeric State of Avian Sarcoma Virus Integrase

Bao¹,

Wang²,

Miller³

et al. 2003

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Presteady-state Analysis of Avian Sarcoma Virus Integrase

Bao

Skalka

Wong

2002

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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...

Regulation of Replication Protein A Functions in DNA Mismatch Repair by Phosphorylation

Guo

Zhang

Yuan

et al. 2006

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Replication protein A (RPA) is involved in multiple stages of DNA mismatch repair (MMR); however, the modulation of its functions between different stages is unknown. We show here that phosphorylation likely modulates RPA functions during MMR. Unphosphorylated RPA initially binds to nicked heteroduplex DNA to facilitate assembly of the MMR initiation complex. The unphosphorylated protein preferentially stimulates mismatch-provoked excision, possibly by cooperatively binding to the resultant single-stranded DNA gap. The DNA-bound RPA begins to be phosphorylated after extensive excision, resulting in severalfold reduction in the DNA binding affinity of RPA. Thus, during the phase of repair DNA synthesis, the phosphorylated RPA readily disassociates from DNA, making the DNA template available for DNA polymerase ␦-catalyzed resynthesis. These observations support a model of how phosphorylation alters the DNA binding affinity of RPA to fulfill its differential requirement at the various stages of MMR.