Reconstitution of Concerted DNA Integration with Purified Components

Hindmarsh, Patrick L.; Leis, Jonathan

doi:10.1016/s0065-3527(08)60308-5

Cited by 56 publications

(60 citation statements)

References 40 publications

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“…3 H-L-731,988 (4-[1-(4-f luorobenzyl)pyrrole-2-yl-2,4-diketobutanoic acid) ( 3 H-I) was prepared by catalytic tritiation of the iodopyrrole (24). § The synthesis of compounds I and II [ 3 H-II, 4-(3-benzylphenyl)-2,4-dioxo-butyric acid] was as described (25).…”

Section: Methodsmentioning

confidence: 99%

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Diketo acid inhibitor mechanism and HIV-1 integrase: Implications for metal binding in the active site of phosphotransferase enzymes

Grobler

Stillmock²,

Hu³

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

378

325

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The process of integrating the reverse-transcribed HIV-1 DNA into the host chromosomal DNA is catalyzed by the virally encoded enzyme integrase (IN). Integration requires two metal-dependent reactions, 3 end processing and strand transfer. Compounds that contain a diketo acid moiety have been shown to selectively inhibit the strand transfer reaction of IN in vitro and in infected cells and are effective as inhibitors of HIV-1 replication. To characterize the molecular basis of inhibition, we used functional assays and binding assays to evaluate a series of structurally related analogs. These studies focused on investigating the role of the conserved carboxylate and metal binding. We demonstrate that an acidic moiety such as a carboxylate or isosteric heterocycle is not required for binding to the enzyme complex but is essential for inhibition and confers distinct metaldependent properties on the inhibitor. Binding requires divalent metal and resistance is metal dependent with active site mutants displaying resistance only when the enzymes are evaluated in the context of Mg 2؉ . The mechanism of action of these inhibitors is therefore likely a consequence of the interaction between the acid moiety and metal ion(s) in the IN active site, resulting in a functional sequestration of the critical metal cofactor(s). These studies thus have implications for modeling active site inhibitors of IN, designing and evaluating analogs with improved efficacy, and identifying inhibitors of other metal-dependent phosphotransferases.A n essential step in HIV replication is the integration of the reverse-transcribed viral genome into host chromosomal DNA by the virally encoded integrase (IN) protein (1-3). Integration is required for efficient long terminal repeat-driven transcription of the provirus for the production of viral proteins and RNA progeny. IN represents an important chemotherapeutic target, as its inactivation, either by mutagenesis or inhibition, blocks productive infection by HIV-1 (4-7).Integration is carried out in the cell in a series of distinct steps (8-10). First, IN cleaves the two terminal nucleotides from each 3Ј end of the viral DNA. The 3Ј processing reaction is carried out concurrently with or soon after reverse transcription in the cytoplasm. In the second step, strand transfer, IN catalyzes staggered nicking of the target chromosomal DNA and joining of each 3Ј end of the viral DNA to the 5Ј ends of the host DNA. Strand transfer is temporally and spatially separated from 3Ј processing and occurs after transport of the preintegration complex from the cytoplasm into the nucleus.Divalent metals such as Mg 2ϩ or Mn 2ϩ are required for both 3Ј processing and strand transfer and for the assembly of IN onto specific viral donor DNA to form a complex competent to carry out either function (11)(12)(13) (18), L-731,988 and related DKAs inhibit integration and viral replication in cell culture (7). Mutations that confer resistance to DKAs have been identified and map to IN residues adjacent to D64 and E152. The in...

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Section: Methodsmentioning

confidence: 99%

“…A n essential step in HIV replication is the integration of the reverse-transcribed viral genome into host chromosomal DNA by the virally encoded integrase (IN) protein (1)(2)(3). Integration is required for efficient long terminal repeat-driven transcription of the provirus for the production of viral proteins and RNA progeny.…”

mentioning

confidence: 99%

Diketo acid inhibitor mechanism and HIV-1 integrase: Implications for metal binding in the active site of phosphotransferase enzymes

Grobler

Stillmock²,

Hu³

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

378

325

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“…Indeed, characterization of retroviral integration processes has shown only a partial dependence on host cell factors, suggesting that viral factors can also be involved in the integration. [18][19][20] Thus, some viral DNA integration events appear to be identical in nature to any type of foreign DNA integration (for example SV40 DNA), while those depending on viral-encoded machinery are clearly different. These types of virus-specific events will not be considered here, but it should be noted that many of the mechanisms involved in the illegitimate integration of foreign DNA could also be involved, at one point or another, in viral DNA integration.…”

Section: Introductionmentioning

confidence: 99%

Illegitimate DNA integration in mammalian cells

2003

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Foreign DNA integration is one of the most widely exploited cellular processes in molecular biology. Its technical use permits us to alter a cellular genome by incorporating a fragment of foreign DNA into the chromosomal DNA. This process employs the cell's own endogenous DNA modification and repair machinery. Two main classes of integration mechanisms exist: those that draw on sequence similarity between the foreign and genomic sequences to carry out homology-directed modifications, and the nonhomologous or 'illegitimate' insertion of foreign DNA into the genome. Gene therapy procedures can result in illegitimate integration of introduced sequences and thus pose a risk of unforeseeable genomic alterations. The choice of insertion site, the degree to which the foreign DNA and endogenous locus are modified before or during integration, and the resulting impact on structure, expression, and stability of the genome are all factors of illegitimate DNA integration that must be considered, in particular when designing genetic therapies.

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“…It is a key step in retroviral infection and subsequent persistence of the retroviral clone during replication of the host cell (1,2). Genomic integration of foreign DNA also has more benign functions.…”

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confidence: 99%

The SET domain protein Metnase mediates foreign DNA integration and links integration to nonhomologous end-joining repair

Lee

Oshige

Durant

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

150

343

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The molecular mechanism by which foreign DNA integrates into the human genome is poorly understood yet critical to many disease processes, including retroviral infection and carcinogenesis, and to gene therapy. We hypothesized that the mechanism of genomic integration may be similar to transposition in lower organisms. We identified a protein, termed Metnase, that has a SET domain and a transposase͞nuclease domain. Metnase methylates histone H3 lysines 4 and 36, which are associated with open chromatin. Metnase increases resistance to ionizing radiation and increases nonhomologous end-joining repair of DNA doublestrand breaks. Most significantly, Metnase promotes integration of exogenous DNA into the genomes of host cells. Therefore, Metnase is a nonhomologous end-joining repair protein that regulates genomic integration of exogenous DNA and establishes a relationship among histone modification, DNA repair, and integration. The data suggest a model wherein Metnase promotes integration of exogenous DNA by opening chromatin and facilitating joining of DNA ends. This study demonstrates that eukaryotic transposase domains can have important cell functions beyond transposition of genetic elements.DNA repair ͉ histone methylation

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Reconstitution of Concerted DNA Integration with Purified Components

Cited by 56 publications

References 40 publications

Diketo acid inhibitor mechanism and HIV-1 integrase: Implications for metal binding in the active site of phosphotransferase enzymes

Diketo acid inhibitor mechanism and HIV-1 integrase: Implications for metal binding in the active site of phosphotransferase enzymes

Illegitimate DNA integration in mammalian cells

The SET domain protein Metnase mediates foreign DNA integration and links integration to nonhomologous end-joining repair

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