Figure 1.
DNA substrates used for crosslinking to ASV IN.
The indicated numbering is as used throughout the manuscript. The Y-mer strand Y4 has non-wild type bases at positions 20 and 21 in order to increase the stability of this arm of the substrate. DNA strands in the Y-mer substrate are colored for identification as in Figures 3–6. The host portion of strand Y4 is colored in lighter shade of blue to distinguish the host sequence from the viral sequence after covalent joining to viral DNA. In linear and Ymer DNAs, the conserved adenine preceding the scissile phosphate is shown in bold.
Figure 2.
Structure-based sequence alignment of full-length ASV, HIV-1, and PFV IN proteins.
ASV IN numbering is shown above the sequences and the structural elements are marked in green; PFV IN numbering and structural elements (black) are shown below. Numbering for HIV-1 IN is shown at the beginning and end of the lines only. The conserved amino acids, including the catalytic ASV IN residues Asp121 and Glu157, are red and boxed. Triangles mark residues that were changed to cysteines in ASV IN: red for the amino acids in the active site, cyan for other residues in the CCD, and magenta for the amino acids in the CTD. The structure of the ASV IN CCD and CTD (PDB code 1COM) with the location of the introduced cysteines is shown in the upper right corner, with the colors corresponding to the scheme described above.
Figure 3.
A comparison of all the IN-DNA contact points that have been determined experimentally or modeled for the NTD IN.
The amino acid residues are presented in black, unless a particular residue comes from specified IN monomer in PFV intasome structure as in Refs. [6], [7]. Specific residues shown to interact with DNA that are either in good correlation with the PFV structural results or do not contradict them are bolded. # -this amino acid is in contact with DNA, but the nucleotide is not determined. (G377) - The amino acid residues in parentheses indicate structural analogs to the ones implicated in DNA binding by experimental data. Nucleotides from different strands of DNA substrates are labeled by colors corresponding to the scheme used in Figure 1 and noted above. “G5{15}” - In this example and throughout Figures 3, 4, 5, 6 the nucleotide numbers correspond to the numbering scheme shown in Figure 1. The numbers in the curly brackets are as in the structure of PFV IN and the model of HIV-1 IN [6], [7]. If listed, the letter designating a nucleotide comes from the original data. All reported contacts are references to original publications with numbers in brackets; our data are marked with asterisks (e.g. A3*).
Figure 4.
A comparison of all the IN-DNA contact points that have been determined experimentally or modeled for the CCD IN.
For details, see legend to Figure 3.
Figure 5.
A comparison of all the IN-DNA contact points that have been determined experimentally or modeled for the CCD IN (continued).
For details, see legend to Figure 3.
Figure 6.
A comparison of all the IN-DNA contact points that have been determined experimentally or modeled for the CTD IN.
For details, see legend to Figure 3.
Table 1.
Substitutions in the ASV IN derivatives and their enzymatic activities (crosslinkers placed at residues that are bold).
Figure 7.
Cel 1-based localization of the crosslinking sites on the Y-mer DNA.
(A) The method for detecting specific UV-mediated crosslinks in Ymer DNA is outlined. (B) Cel 1 cleavage of the photocrosslinked complex of IN I146C. Products from Y-mer DNAs labeled at the 5′-end of strand 3 or 4 (marked above each lane) are shown. The filled arrows point to prominent Cel 1 products, indicative of a bulky adduct at the conserved viral CA dinucleotide in strand 4. Open arrows mark the position of non-cleaved substrate strands. (C) Cel 1 cleavage of various photocrosslinked complexes of Cys-modified derivatives of IN with Y-mer DNA labeled at strands 2, 3, or 4. Numbers above the gels indicate which DNA strand in the Y-mer was labeled. Open arrows mark the position of non-cleaved substrate strands. Numbers to the left of the gel indicate length in nucleotides, and arrows to the right mark the positions of adducts of IN with DNA. In both B and C, products were separated by denaturing gel electrophoresis and then visualized with a PhosphorImager. (D) Y-mer DNA sequence with positions of preferred crosslinking detected by Cel1 indicated for each IN derivative by red (I146C), green (R244C) and teal (S124C) arrows; (E) 3-D model of the Y-mer DNA with positions of preferred crosslinking detected by Cel1 indicated for each IN derivative by green (C244), red (C146) and teal (C124) dots.
Table 2.
Preferred position for photocrosslinking of modified IN Cys derivatives to a Y-mer DNA substrate.
Table 3.
A comparative summary of IN-DNA S-S crosslinking with mixed disulfide–modified substrates.
Figure 8.
Design of modified 3′-end adenine.
A nucleotide at the 3′-end of DNA in TN5 transposase structure is shown in blue, morpholino adenosine analog in yellow, nucleotides with modifications on the C3′ and on C2′ of ribose,are shown in pink and gray respectively. Catalytic residues are shown in blue for transposase and in green for ASV IN. Substituted catalytic residues D64C and E157C are shown in magenta.
Figure 9.
S-S crosslinking of the ASV IN active site derivatives to modified linear dsDNA substrates.
Sample Coomassie–stained polyacrylamide gels with IN-DNA crosslinks. pH indicates pH-induced crosslinking; DTNB, DTNB-mediated crosslinking; lanes labeled P-SS correspond to crosslinking to DNA carrying thiol modification on the 3′ phosphate, lanes labeled M3 correspond to crosslinking to DNA carrying thiol modification on the morpholino adenosine analog. The lane marked Neg represents samples with no DNA. 2IN, IN+DNA and 1IN designate dimeric IN bands, adduct bands and monomeric IN bands, respectively. Molecular weight marker lanes are not shown as IN-DNA the monomer (lower) and dimer (upper) bands provide internal calibration. The figure shows the results only for full length INs. Panel A shows crosslinking to the C23S/C125S/E157C/F199K ASV IN derivative (labeled Cys157 after the key mutation); panel B to C23S/C125S/E157C/F199K/W259A (labeled Cys157/Ala259), and panel C to D64C/F199K (labeled Cys64).
Figure 10.
Structural interpretation of crosslinking data for ASV IN.
(A) Superposition of a section of the DNA complex of PFV IN (cartoon) containing the two viral DNAs (sticks) with the individual CCD and CTD domains of ASV IN. Individual protomers of the PFV IN tetramer are colored gray and yellow, with Asn348 shown as sticks in corresponding colors. CCDs of ASV IN are magenta and blue and one CTD of ASV is shown in magenta, with Arg244 shown in sticks in corresponding colors. The linker region of PFV is shown in yellow. Nucleotides 10–12 of the non-cleaved viral DNA strand are shown in dark violet, nucleotides 11 and 12 of the cleaved viral DNA strand are shown in magenta, nucleotide 7 of the non-cleaved viral DNA strand is shown in cyan. (B) Superposition of the CCDs of ASV IN (blue) onto PFV IN (gray). The viral DNA after the integration step is shown in green and the host target DNA in orange. Nucleotides 3 and 8 on the host DNA are red and the substituted residue S124C is shown as sticks. (C) An analogous superposition, with viral DNA in red (non-cleaved strand) and pink (processed strand). A flexible loop contains the substituted residue I146C. Nucleotide 2 on the non-cleaved strand and the first nucleotide on the 3′-end of the cleaved strand are shown in green.