Substrate Recognition by Retroviral Integrases

Katzman, Michael; Katz, Richard A.

doi:10.1016/s0065-3527(08)60307-3

Cited by 42 publications

(50 citation statements)

References 123 publications

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“…However, as previously reported (22), there were "hot spots" for integration. In several instances we detected integration at the same site to the base (in either orientation) (Tables I-VIII).…”

Section: Location Of Integrants In the Acceptorsupporting

confidence: 67%

Changes in the Mechanism of DNA Integration in Vitro Induced by Base Substitutions in the HIV-1 U5 and U3 Terminal Sequences

Brin

Leis

2002

Journal of Biological Chemistry

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We have reconstituted concerted human immunodeficiency virus type 1 (HIV-1) integration with specially designed mini-donor DNA, a supercoiled plasmid acceptor, purified bacterial-derived HIV-1 integrase (IN), and host HMG-I(Y) protein (Hindmarsh, P., Ridky, T., Reeves, R., Andrake, M., Skalka, A. M., and Leis, J. (1999) J. Virol. 73, 2994 -3003). Integration in this system is dependent upon the mini donor DNA having IN recognition sequences at both ends and the reaction products have all of the features associated with integration of viral DNA in vivo. Using this system, we explored the relationship between the HIV-1 U3 and U5 IN recognition sequences by analyzing substrates that contain either two U3 or two U5 terminal sequences. Both substrates caused severe defects to integration but with different effects on the mechanism indicating that the U3 and the U5 sequences are both required for concerted DNA integration. We have also used the reconstituted system to compare the mechanism of integration catalyzed by HIV-1 to that of avian sarcoma virus by analyzing the effect of defined mutations introduced into U3 or U5 ends of the respective wild type DNA substrates. Despite sequence differences between avian sarcoma virus and HIV-1 IN and their recognition sequences, the consequences of analogous base pair substitutions at the same relative positions of the respective IN recognition sequences were very similar. This highlights the common mechanism of integration shared by these two different viruses.Integration of retroviral DNA is an obligatory step in viral replication. Integration is catalyzed by the viral encoded enzyme, integrase (IN), 1 which brings the ends of a linear viral DNA together and inserts them into the host chromosome in a concerted reaction (see Ref. 1 for a review). Cell proteins belonging to the HMG-I(Y) family stimulate the reaction (2, 3). The sites of integration are widely distributed in the target DNA. Short duplications of the cell DNA are introduced at the insertion site, the size of which is dictated by IN. For HIV-1 and ASV the size of the duplications are five and six base pairs, respectively. During this process, two base pairs are lost from the ends of the viral LTRs.The properties of concerted DNA integration have been reconstituted in vitro using purified HIV-1 (3, 4) or ASV (3, 5-11) IN and MgCl 2 . The donor DNAs contain only 20 base pairs for HIV-1 or 15 base pairs for ASV derived from the ends of the LTRs, respectively. These viral DNA end sequences correspond to the nearly perfect inverted repeats that define the relationships between the U3 and U5 RNA ends. The inverted repeat for RSV is 12 of 15, while that for HIV-1 is 12 of 20. A comparison of the RSV and HIV-1 IN recognition sequences indicates that they are unique. The only common feature is the presence of a conserved CA dinucleotide at positions 3 and 4 from the terminus. Short oligodeoxynucleotide duplexes representing the ends of HIV-1 U5 LTR are more efficient substrates for IN processing (12-15) and strand transf...

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Section: Location Of Integrants In the Acceptorsupporting

confidence: 67%

Changes in the Mechanism of DNA Integration in Vitro Induced by Base Substitutions in the HIV-1 U5 and U3 Terminal Sequences

Brin

Leis

2002

Journal of Biological Chemistry

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“…Three point mutants were constructed, N210S and D248S, which alters known conserved essential SET domain amino acids, and D490S, which alters a known essential conserved amino acid in the Transposase domain (32)(33)(34)(35)(36). Three mutants were also generated that altered three or four amino acids in these same regions that harbored these essential residues: the NHSC at 210-213 was altered to AAAA, the YDY at 247-249 to AAA, and the YDN at 489-491 to AAA.…”

Section: Resultsmentioning

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.

147

341

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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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“…4B) and to bread wheat (T. aestivum), timothy (Phleum pretense), cultivars of turnip rape (Brassica rapa), and canola (B. napus) (data not shown) and observed levels of polymorphism that are generally higher than those obtained with families of protein-coding, autonomous retrotransposons. Integrase, encoded by retrotransposons, creates target site duplications (TSDs) as it inserts new elements (31). Hence, detection of TSDs flanking genomic copies provides evidence for retrotransposition.…”

Section: S Sequences In Cassandra Ltrsmentioning

confidence: 99%

Cassandraretrotransposons carry independently transcribed 5S RNA

Kalendar

Tanskanen

Chang

et al. 2008

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

123

141

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We report a group of TRIMs (terminal-repeat retrotransposons in miniature), which are small nonautonomous retrotransposons. These elements, named Cassandra, universally carry conserved 5S RNA sequences and associated RNA polymerase (pol) III promoters and terminators in their long terminal repeats (LTRs). They were found in all vascular plants investigated. Uniquely for LTR retrotransposons, Cassandra produces noncapped, polyadenylated transcripts from the 5S pol III promoter. Capped, read-through transcripts containing Cassandra sequences can also be detected in RNA and in EST databases. The predicted Cassandra RNA 5S secondary structures resemble those for cellular 5S rRNA, with high information content specifically in the pol III promoter region. Genic integration sites are common for Cassandra, an unusual feature for abundant retrotransposons. The 5S in each LTR produces a tandem 5S arrangement with an inter-5S spacing resembling that of cellular 5S. The distribution of 5S genes is very variable in flowering plants and may be partially explained by Cassandra activity. Cassandra thus appears both to have adapted a ubiquitous cellular gene for ribosomal RNA for use as a promoter and to parasitize an as-yet-unidentified group of retrotransposons for the proteins needed in its lifecycle.pol III ͉ genome evolution ͉ transcription ͉ transposable element R etrotransposons, excepting SINEs (short interspersed nuclear elements) and LINEs (long interspersed nuclear elements), resemble retroviruses in their structure and intracellular life cycle. They are ubiquitous in the genomes of plants, animals, and fungi and account for Ͼ50% of large plant genomes (1, 2). Their life cycle comprises transcription of genomic copies, translation of their encoded proteins, packaging of the transcripts into virus-like particles, reverse transcription, and targeting of the cDNA copy to the nucleus for integration into the genome (3, 4). The transcriptional signals for RNA polymerase II (pol II) are found in the long terminal repeats (LTRs) at either end of the element, flanking the priming sites for reverse transcription and the coding domain specifying the proteins needed for replication and integration [supporting information (SI) Fig. S1].In addition to the classical retrotransposons, several well conserved nonautonomous groups have been discovered that lack all or part of their coding capacity (5). The BARE2 elements cannot express the capsid protein GAG (6), and Morgane lacks most of its coding capacity (7). The TRIM (terminal repeat retrotransposon in miniature) and LARD (large retrotransposon derivative) elements (Fig. S1) entirely lack reading frames for retrotransposon proteins (8-12). The TRIM elements are composed of 100-to 250-bp LTRs, priming sites for reverse transcriptase, and a small intervening segment. Evidence for past mobility suggests that they are activated by transcomplementation (10). These have been found in at least 13 species from four plant families (9, 10).Here, we describe a group of TRIM elements, which w...

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Substrate Recognition by Retroviral Integrases

Cited by 42 publications

References 123 publications

Changes in the Mechanism of DNA Integration in Vitro Induced by Base Substitutions in the HIV-1 U5 and U3 Terminal Sequences

Changes in the Mechanism of DNA Integration in Vitro Induced by Base Substitutions in the HIV-1 U5 and U3 Terminal Sequences

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

Cassandraretrotransposons carry independently transcribed 5S RNA

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