ABSTRACT
MOTIVATION: Direct recognition, or direct readout, of DNA bases by a DNA-binding protein involves amino acids that interact directly with features specific to each base. Experimental evidence also shows that in many cases the protein achieves partial sequence specificity by indirect recognition, i.e., by recognizing structural properties of the DNA. (1) Could threading a DNA sequence onto a crystal structure of bound DNA help explain the indirect recognition component of sequence specificity? (2) Might the resulting pure-structure computational motif manifest itself in familiar sequence-based computational motifs? RESULTS: The starting structure motif was a crystal structure of DNA bound to the integration host factor protein (IHF) of E. coli. IHF is known to exhibit both direct and indirect recognition of its binding sites. (1) Threading DNA sequences onto the crystal structure showed statistically significant partial separation of 60 IHF binding sites from random and intragenic sequences and was positively correlated with binding affinity. (2) The crystal structure was shown to be equivalent to a linear Markov network, and so, to a joint probability distribution over sequences, computable in linear time. It was transformed algorithmically into several common pure-sequence representations, including (a) small sets of short exact strings, (b) weight matrices, (c) consensus regular patterns, (d) multiple sequence alignments, and (e) phylogenetic trees. In all cases the pure-sequence motifs retained statistically significant partial separation of the IHF binding sites from random and intragenic sequences. Most exhibited positive correlation with binding affinity. The multiple alignment showed some conserved columns, and the phylogenetic tree partially mixed low-energy sequences with IHF binding sites but separated high-energy sequences. The conclusion is that deformation energy explains part of indirect recognition, which explains part of IHF sequence-specific binding.
Subject(s)
Algorithms , DNA, Bacterial/chemistry , DNA-Binding Proteins/chemistry , Escherichia coli Proteins/chemistry , Integration Host Factors/chemistry , Models, Chemical , Models, Molecular , Sequence Analysis, DNA/methods , Amino Acid Sequence , Binding Sites , Macromolecular Substances , Models, Statistical , Molecular Sequence Data , Protein Binding , Sequence Alignment/methods , Structure-Activity RelationshipABSTRACT
BACKGROUND: Integration of the HIV genome by integrase is absolutely required for productive infection. OBJECTIVE: To determine the role of natural selection on HIV integrase biology. DESIGN: To study the activities of HIV integrases from a limited panel of North American clinical isolates from HIV-infected patients and to compare these proteins with integrases from two laboratory adapted reference strains (HI(VIIIRF) and HIV(NL4--3)). METHODS: HIV was isolated and the particle-associated RNA was reverse transcribed and sequenced. Replication kinetics of molecularly cloned viruses containing each variant integrase were studied in tissue culture. The mutant integrase proteins were expressed, purified and specific activities of the enzymes were derived for both 3' end-processing and disintegration reactions. RESULTS: Despite 3--5% variability in integrase at the amino acid level, viruses showed no statistically significant differences in growth kinetics compared with the reference HIV(NL4--3) virus and only minor differences were observed in 3' end-processing and disintegration activities. All integrase proteins demonstrated similar sensitivity to an integrase inhibitor l-chicoric acid. CONCLUSIONS: These results demonstrate that integrase genes derived from HIV-infected individuals can differ from reference sequences but these mutations do not result in loss of function, including susceptibility to an integrase inhibitor; therefore, integrase remains an attractive target for antiviral drug design, as mutability appears to be restricted by function.
