Does tryptophan intercalate in DNA? A comparative study of peptide binding to alternating and non-alternating A.cntdot.T sequences

The nucleocapsid protein of simian immunodeficiency virus (SIV) NCp8 has two copies of conserved sequences (termed zinc fingers, ZF) of 14 amino acids with 4 invariant residues (CCHC) that coordinate Zn(II). Each of its two ZFs has a Trp residue. A significant quenching of NCp8 Trp fluorescence was seen in nucleic acid complexes, suggesting stacking of the indole ring with nucleobases and the simultaneous involvement of both ZFs in the binding process. Both ZFs contribute to the nucleic acid binding free energy of NCp8, albeit in a not additive manner. NCp8 exhibited a base preference analogous to that of NCp7: G ϳ I > T > U > C > A. Alternating base sequences that bind HIV-1 NCp7 in a sequence-specific manner were also bound selectively by NCp8. Specific sequence recognition required at least five bases and the presence of bound Zn(II). The two ZFs account for the net displacement of 3 out of 4 sodium ions upon binding (2 by the first and one by the second finger), and for most (85%) of the hydrophobic stabilization in complex formation. Based on the sequence and functional similarity of SIV NCp8 and HIV-1 NCp7, and using available structural information for free and oligonucleotide bound NCp7, we propose a structural model for NCp8-oligonucleotide complexes.

Section: Resultssupporting

confidence: 49%

Nucleic Acid Binding Properties of the Simian Immunodeficiency Virus Nucleocapsid Protein NCp8

Urbaneja

McGrath

Kane

et al. 2000

“…4B), at a position where the tryptophan indole could make specific contacts with DNA nucleobases. Because a tryptophyl residue can intercalate specifically with DNA (48,49), Trp 324 is a potential determinant for sequence specificity. In contrast, in other phages like T4 (that has Glu 404 in lieu of Trp 324 ), although a pac site has been reported (50), the first cut does not strictly occur in the proximity of a unique pac sequence (51).…”

Section: Discussionmentioning

confidence: 99%

Structure of P22 Headful Packaging Nuclease

Roy

Cingolani

2012

Background:The headful nuclease is essential for genome processing during viral DNA packaging. Results: The crystal structure of P22 nuclease reveals a seven-stranded ␤-sheet core with two magnesium ions poised for catalysis. Conclusion: P22 headful nuclease is active only in the context of the large terminase. Significance: The C-terminal headful nuclease is activated by intramolecular cross-talk with the N-terminal ATPase domain.

“…Effect of Peptide Sequence and Reaction Conditions on Nonelectrostatic Interactions-It has been previously reported that short peptides of polylysine containing Trp residues bind to polynucleotides primarily by electrostatic interactions (18,19,(25)(26)(27). To investigate whether these differences with our observations were a consequence of particular peptide sequences or different reaction conditions, we compared the binding of KWK to poly(U) under our conditions at pH 7.4 with the same reaction at pH 5.8, which has been previously well studied (19).…”

Section: Preparation and Characterization Of Peptides And Polynucleotmentioning

confidence: 99%

“…Trp and polynucleotides were purchased from Sigma. Polynucleotides were exhaustively dialyzed against reaction buffer, and their concentrations were determined in 0.1 M Tris acetate, pH 7.4, at 25°C from published extinction coefficients (18,19). Solutions were prepared with filtered deionized water (Milli-Q, Millipore Corp.).…”

mentioning

confidence: 99%

Strandedness Discrimination in Peptide-Polynucleotide Complexes

Johnson

Mazarguil

Lopez

1996

Preferential binding to single-or double-stranded nucleic acids is important for the activity of many proteins that process RNA and DNA. We have investigated the mechanism of strandedness discrimination with peptides derived from the putative DNA-binding domain of the RecA protein, a bacterial recombinase that modulates its affinity for single-stranded DNA by means of ATP binding and hydrolysis. Contributions of electrostatic and non-electrostatic interactions to binding of these peptides with polynucleotides were evaluated by fluorescence spectroscopy as a function of salt concentration and peptide charge. Binding of these peptides to single-and double-stranded nucleic acids was dominated by non-electrostatic interactions. Small electrostatic contributions selectively enhanced peptide complexation with single-stranded nucleic acids. Similar results were observed in control experiments carried out with tripeptides containing charged and aromatic amino acid residues. It was possible to modify the strandedness preference of peptide-polynucleotide complexes by changing electrostatic contributions to the binding free energy. These observations suggest a mechanism whereby some proteins that interact with DNA or RNA might determine and regulate their relative affinity for single-and double-stranded nucleic acids.Preferential binding to single-or double-stranded polynucleotides is characteristic of numerous proteins involved in DNA replication, transcription, DNA repair, and homologous recombination (1). Single-stranded binding proteins transiently stabilize single-stranded polynucleotides without NTP hydrolysis (2, 3). Helicases and recombinases may switch strandedness specificity by NTP-dependent mechanisms (4, 5). These proteins generally bind to nucleic acids cooperatively or as multimeric complexes. It is important to understand how these proteins recognize single-and double-stranded polynucleotides and how they change strandedness preference.Protein sequence motifs that recognize single-stranded DNA generally include both positively charged residues that can interact electrostatically with phosphate groups in the polynucleotide backbone and aromatic amino acids that can associate with nucleic acid bases via non-electrostatic interactions (2, 3, 6, 7). The L2 loop, a putative DNA-binding domain of the RecA protein, has charged and aromatic amino acid residues characteristic of single-stranded DNA-binding proteins; these general features are found in many recombinases (Fig. 1a) (8 -10). Genetic and biochemical experiments show that point mutations in this loop disrupt RecA protein function in vivo and in vitro (11)(12)(13)(14)(15) and modify the affinity of the RecA protein for single-stranded DNA (15). This loop is not resolved in the crystal structure of the RecA-ADP complex (16), which suggests that it may be flexible in the absence of DNA and that, at least to a certain extent, it might interact with DNA independently of the protein scaffolding. Hence, a peptide corresponding to the L2 loop may reproduce some o...