Document 0709
 DOCN  M9590709
 TI    In vitro activities of hiv-1 integrase/e. coli lexa fusion proteins.
 DT    9509
 AU    Chow SA; Goulaouic H; Department of Molecular and Medical Pharmacology,
       UCLA School of; Medicine, Los Angeles, CA
 SO    NIH Conf Retroviral Integrase. 1995 Jan 19-20;:(Session II, speakers'
       abstracts - unpaged). Unique Identifier : AIDSLINE AIDS/95920010
 AB    One salient feature of retroviral integration is that many sites on the
       host DNA can be used as targets. The integration process, however, is
       not entirely random since there is a wide variation in integration
       frequency among individual target sites. The mechanism for selecting DNA
       sites for integration is not fully understood. It is also not clear
       which integrase domain is involved in recognition of target DNA, though
       results obtained from several retroviral integrases show that the
       C-terminal domain is capable of binding to DNA without sequence
       specificity. An earlier report showed that integration of viral DNA
       mediated by a fusion protein consisting of HIV-1 integrase and phage
       lambda repressor is biased toward DNA containing a lambda operator
       sequence. In this study, we constructed a protein that has the E. coli
       LexA protein fused to the C-terminus of the HIV-1 integrase
       (IN1-288/LA). As an attempt to increase the dependence of the fusion
       protein of the LexA component for target DNA recognition, we also
       constructed two fusion proteins in which the C-terminus of the HIV-1
       integrase was truncated by 54 (IN1-234/LA) or 102 amino acids (IN1-
       186/LA). The fusion proteins, which contain a six-histidine tag at the
       N-terminus, were overexpressed in E. coli and purified by
       nickel-chelating affinity chromatography. The catalytic activities of
       the fusion proteins were tested by 3-end processing, 3'-end joining, and
       disintegration assays. IN1-288/LA behaved as the wild-type integrase and
       was active in all three assays; whereas the IN1-186/LA was completely
       inactive. IN1-234/LA could carry out disintegration, but had no
       detectable 3'-end processing and 3'-end joining activities using a
       blunt-ended oligonucleotide substrate. However, using a pre-processed
       substrate, a weak 3-end joining activity was detected. To examine the
       effect of the LexA component on the integration frequency of IN1-288/LA,
       we inserted a LexA binding sequence into the Kpn I site of the plasmid
       pBluescript KS II+. The plasmid DNA, after cleavage with Mbo II to
       generate multiple fragments, was used as target DNA in a strand transfer
       assay containing HIV U5 oligonucleotide as the donor DNA. We found that
       the donor DNA was preferably integrated into the fragment containing the
       LexA sequence. No bias was observed when the fusion protein was replaced
       by the wild-type integrase, or when LexA protein was added in the
       reaction containing IN1- 288/LA. The integration patterns of the
       wild-type integrase and the fusion proteins IN1-288/LA and IN1-234/LA
       were also determined using a PCR-based assay. The integration patterns
       of IN1-288/LA and IN1-234/LA were similar to each other but were
       different from that of the wild-type. Compared to the wild-type, the
       region corresponding to the LexA sequence was devoid of any integration,
       and a majority of the integration events occurred near the regions
       flanking the LexA sequence. The results show that the selection of DNA
       sites for integration can be affected by fusing integrase to
       sequence-specific DNA binding protein.
 DE    Bacterial Proteins/GENETICS  Catalysis  Cloning, Molecular  DNA
       Nucleotidyltransferases/GENETICS/*METABOLISM  DNA, Viral/GENETICS
       DNA-Binding Proteins/METABOLISM  Escherichia coli/*GENETICS
       HIV-1/*ENZYMOLOGY  Oligonucleotides/METABOLISM  Protein Processing,
       Post-Translational  Recombinant Fusion Proteins/GENETICS/*METABOLISM
       Repressor Proteins/GENETICS  Virus Integration/GENETICS  MEETING
       ABSTRACT

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).