Characterization of a DNA binding domain in the C-terminus of HIV-1 integrase by deletion mutagenesis.
AUTOR(ES)
Woerner, A M
RESUMO
The integrase (IN) protein of human immunodeficiency virus type 1 (HIV-1) catalyzes site-specific cleavage of 2 bases from the viral long terminal repeat (LTR) sequence yet it binds DNA with little DNA sequence specificity. We have previously demonstrated that the C-terminal half of IN (amino acids 154-288) possesses a DNA binding domain. In order to further characterize this region, a series of clones expressing truncated forms of IN as N-terminal fusion proteins in E.coli were constructed and analyzed by Southwestern blotting. Proteins containing amino acids 1-263, 1-248 and 170-288 retained the ability to bind DNA, whereas a protein containing amino acids 1-180 showed no detectable DNA binding. This defines a DNA binding domain contained within amino acids 180-248. This region contains an arrangement of 9 lysine and arginine residues each separated by 2-4 amino acids (KxxxKxxxKxxxxRxxxRxxRxxxxKxxxKxxxK), spanning amino acids 211-244, which is conserved in all HIV-1 isolates. A clone expressing full-length IN with a C-terminal fusion of 16 amino acids was able to bind DNA comparably to a cloned protein with a free C-terminus, and an IN-specific monoclonal antibody which recognizes an epitope contained within amino acids 264-279 was unable to block DNA binding, supporting the evidence that a region necessary for binding lies upstream of amino acid 264.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=331452Documentos Relacionados
- Regulation of site-specific recombination by the C-terminus of λ integrase
- A DNA binding domain is contained in the C-terminus of wild type p53 protein.
- Characterization of the bacteriophage lambda excisionase (Xis) protein: the C-terminus is required for Xis-integrase cooperativity but not for DNA binding.
- Function of the C-terminus of φ29 DNA polymerase in DNA and terminal protein binding
- In vitro and in vivo function of the C-terminus of Escherichia coli single-stranded DNA binding protein.