Role of DNA End Distortion in Catalysis by Avian Sarcoma Virus Integrase

Katz, Richard A.; DiCandeloro, Paul; Kukolj, George; Skalka, Anna Marie

doi:10.1074/jbc.m104632200

Cited by 23 publications

(20 citation statements)

References 41 publications

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“…However it must be noted that according to Ref. 42, binding of the avian homolog of HIV integrase to its DNA substrate enhances the accessibility and the subsequent modification of the third thymidine by KMnO 4 . Thus, although integrase disrupts the A/T base pair, it does not protect the heterocyclic base from the modification, thereby ruling out a close contact between the base and the enzyme.…”

Section: Discussionmentioning

confidence: 99%

Probing of HIV-1 Integrase/DNA Interactions Using Novel Analogs of Viral DNA

Agapkina

Smolov

Barbe

et al. 2006

Journal of Biological Chemistry

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The specific activity of the human immunodeficiency virus, type 1 (HIV-1), integrase on the viral long terminal repeat requires the binding of the enzyme to certain sequences located in the U3 and U5 regions at the ends of viral DNA, but the determinants of this specific DNA-protein recognition are not yet completely understood. We synthesized DNA duplexes mimicking the U5 region and containing either 2-modified nucleosides or 1,3-propanediol insertions and studied their interactions with HIV-1 integrase, using Mn 2؉ or Mg 2؉ ions as integrase cofactors. These DNA modifications had no strong effect on integrase binding to the substrate analogs but significantly affected 3-end processing rate. The effects of nucleoside modifications at positions 5, 6, and especially 3 strongly depended on the cationic cofactor used. These effects were much more pronounced in the presence of Mg 2؉ than in the presence of Mn 2؉ . Modifications of base pairs 7-9 affected 3-end processing equally in the presence of both ions. Adenine from the 3rd bp is thought to form at least two hydrogen bonds with integrase that are crucial for specific DNA recognition. The complementary base, thymine, is not important for integrase activity. For other positions, our results suggest that integrase recognizes a fine structure of the sugar-phosphate backbone rather than heterocyclic bases. Integrase interactions with the unprocessed strand at positions 5-8 are more important than interactions with the processed strand for specific substrate recognition. Based on our results, we suggest a model for integrase interaction with the U5 substrate.Following reverse transcription, a DNA copy of the human immunodeficiency virus, type 1 (HIV-1), 2 RNA is integrated into the genome of infected cells. Integration is a prerequisite for viral replication and is catalyzed by the viral enzyme integrase (IN). IN binds to sequences located at the end of U3 and U5 parts of long terminal repeats (LTRs) of viral DNA and catalyzes the trimming, or 3Ј-end processing, of the terminal dinucleotide from the 3Ј-ends of both strands of the DNA. IN then mediates a strand transfer reaction that inserts the viral DNA into the host DNA. During this reaction, IN must bind simultaneously to viral and target DNA. However, IN interacts with these two DNA molecules in different ways as follows: binding to host DNA does not depend directly on host DNA sequence, whereas interaction with the viral DNA is a sequence-specific process. Nevertheless, the U5 and U3 sequences recognized by IN are not exactly identical.Strand transfer and 3Ј-end processing reactions may be carried out in vitro, using recombinant HIV IN, DNA duplexes mimicking U3 or U5 sequences of LTRs, and divalent metal ions, such as Mg 2ϩ or Mn 2ϩ . However, the Mn 2ϩ -and Mg 2ϩ -dependent activities of IN are not equivalent, with lower specificity reported for Mn 2ϩ -dependent IN (1, 2). Moreover, the inhibition of HIV-1 IN by compounds such as ␤-diketo acids, which interact with the active site of HIV-1 IN, is also metal-de...

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Section: Discussionmentioning

confidence: 99%

Probing of HIV-1 Integrase/DNA Interactions Using Novel Analogs of Viral DNA

Agapkina

Smolov

Barbe

et al. 2006

Journal of Biological Chemistry

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“…It is also noteworthy that the RNase H domain of HIV-1 RT exhibits structural similarity with retroviral integrases and Holliday junction endonucleases (31,32). These enzymes have been shown to introduce local distortions in the nucleic acid structure at the scissile bond (17,23,33). It is therefore intriguing to explore whether the same mechanism is employed by the retroviral polymerase for nonspecific hydrolysis of RNA/DNA hybrids.…”

Section: The Rt Positioning On the 3ј Or 5ј Primer Terminus Is Not Crmentioning

confidence: 99%

Pre-existing Distortions in Nucleic Acid Structure Aid Polypurine Tract Selection by HIV-1 Reverse Transcriptase

Kvaratskhelia

Budihas

Grice

2002

Journal of Biological Chemistry

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Precise cleavage at the polypurine tract (PPT)/U3 junction by human immunodeficiency virus type 1 (HIV-1) reverse transcriptase RNase H is critical for generating a correct viral DNA end for subsequent integration. Using potassium permanganate (KMnO 4 ) modification, we have identified a significant distortion in the nucleic acid structure at the HIV-1 PPT/U3 junction in the absence of trans-acting factors. Unusually high reactivity of template thymine ؉1 is detected when the PPT primer is extended by DNA or RNA at its 3 terminus. Chemical footprinting suggests that the extent of base unstacking in the wild-type species is comparable when the ؉1 A:T base pair is replaced by a C:T mismatch. However, reactivity of this template base is diminished after alterations to upstream (rA) 4 :(dT) 4 or (rG) 6 :(dC) 6 tracts. Importantly, there is a correlation between the structural deformation at base pair ؉1 and precise cleavage at the PPT/U3 junction by HIV-1 reverse transcriptase/RNase H. KMnO 4 modification also revealed unusually high reactivity for one of two (dT) 4 : (rA) 4 duplexes upstream of the PPT/U3 junction, suggesting a significant structural distortion within the PPT itself in the absence of the retroviral polymerase. Structural abnormalities in this region are not only essential for resistance of the PPT to hydrolysis but also significantly impact the conformation of the PPT/U3 junction. Our data collectively suggest that the entire PPT sequence contributes to the structural distortion at the PPT/U3 junction, potentially providing a mechanism for its selective processing.In retroviruses, the accuracy with which the (ϩ) strand, polypurine tract (PPT) 1 primer is selected from the RNA-DNA replication intermediate and excised from nascent (ϩ) strand DNA defines the 5Ј long terminal repeat terminus and is critical for production of an integration-competent provirus (1). Because the bulk of the replication intermediate is nonspecifically hydrolyzed, structural features of the PPT (a) render it RNase H-insensitive and (b) control precise cleavage at the junction with adjacent U3 DNA or RNA sequences (1). Although several reports have studied PPT processing relative to alterations in its sequence (2-6) or that of the cognate retroviral polymerase (7-9), the structural basis for this remains elusive. The recent structure of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) bound to a PPTcontaining RNA/DNA duplex has provided important mechanistic insights regarding the PPT resistance to hydrolysis (10). These authors identified an unusual distortion within the (rA) 4 :(dT) 4 stretches of the PPT, i.e. misaligned base pairing between the template and primer nucleotides, suggesting that extension of this deformation into the RNase H active site may confer resistance to hydrolysis. The crystal structure also revealed extensive contacts between the RNase H "primer grip" and the RNA/DNA hybrid. However, because a structure of the complete RNA/DNA duplex PPT in the absence of RT is unavailable, it ...

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“…Total HeLa cell RNA was isolated with the Trizol reagent (Invitrogen) and purified with the RNeasy kit (Qiagen). For the preparation of cDNA probes, 25 g of total RNA was reverse transcribed in the presence of oligo(dT) [12][13][14][15][16][17][18] and aminoallyl-dUTP. The cDNAs were labeled with N-hydroxysuccinimide ester linked to either Cy3 or Cy5 dye, respectively (Amersham Biosciences).…”

Section: Methodsmentioning

confidence: 99%

“…Rather than blocking this process, wrapping of the host DNA into nucleosomes appears to distort the DNA in a way that promotes periodic integration events within the nucleosome, both in vitro and in vivo (27)(28)(29)(30)(31)(32). Recognition of distorted DNA may be a fundamental feature of the IN catalytic mechanism (15,35). Although wrapping of DNA around individual nucleosomes may enhance integration, it is unknown if the preintegration complex has free access to host DNA within higher-order chromatin or if such access depends on other factors.…”

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confidence: 99%

Genome-Wide Analyses of Avian Sarcoma Virus Integration Sites

Narezkina

Taganov

Litwin

et al. 2004

J Virol

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152

156

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The chromosomal features that influence retroviral integration site selection are not well understood. Here, we report the mapping of 226 avian sarcoma virus (ASV) integration sites in the human genome. The results show that the sites are distributed over all chromosomes, and no global bias for integration site selection was detected. However, RNA polymerase II transcription units (protein-encoding genes) appear to be favored targets of ASV integration. The integration frequency within genes is similar to that previously described for murine leukemia virus but distinct from the higher frequency observed with human immunodeficiency virus type 1. We found no evidence for preferred ASV integration sites over the length of genes and immediate flanking regions. Microarray analysis of uninfected HeLa cells revealed that the expression levels of ASV target genes were similar to the median level for all genes represented in the array. Although expressed genes were targets for integration, we found no preference for integration into highly expressed genes. Our results provide a more detailed description of the chromosomal features that may influence ASV integration and support the idea that distinct, virus-specific mechanisms mediate integration site selection. Such differences may be relevant to viral pathogenesis and provide utility in retroviral vector design.Retroviral DNA integration is catalyzed by a viral enzyme, integrase (IN), which nicks the two ends of linear viral DNA and splices them into a site in the host DNA (9, 10). This highly orchestrated reaction produces DNA sequence signatures at the virus-host junctions: the loss of usually 2 bp at the ends of the linear viral DNA and duplication of several base pairs of host DNA at the integration site. However, no gross rearrangements or deletions of either the viral or host DNAs are incurred. The integration reaction can be reproduced in vitro using purified IN and viral and target DNA model substrates. Despite the precision of the integration reaction, there are no strict host DNA sequence requirements. Nevertheless, numerous studies indicate that integration site selection is not likely to be entirely random. For example, various features of host chromosomes have been implicated in influencing integration site selection, including primary DNA sequence (7,14,18), DNA structure (16,20,24), nucleosome structure (27-31), chromatin structure (32,36,37,40), and transcriptional activity (23,33,34,41,43). In addition to the passive influences of chromosomal structure, it has been suggested that retroviral integration could be actively targeted by tethering to specific, chromatin-bound host factors (5). Lastly, the IN proteins from different retroviruses produce unique in vitro integration patterns in naked DNA targets (18). These intriguing but disparate observations have not yet led to a unifying model, and the mechanisms that govern integration site selection in vivo remain obscure.In an infected cell, retroviral DNA is organized in a preintegration complex that in...

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Role of DNA End Distortion in Catalysis by Avian Sarcoma Virus Integrase

Cited by 23 publications

References 41 publications

Probing of HIV-1 Integrase/DNA Interactions Using Novel Analogs of Viral DNA

Probing of HIV-1 Integrase/DNA Interactions Using Novel Analogs of Viral DNA

Pre-existing Distortions in Nucleic Acid Structure Aid Polypurine Tract Selection by HIV-1 Reverse Transcriptase

Genome-Wide Analyses of Avian Sarcoma Virus Integration Sites

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