Figure 1.
Integration into target sequence.
Scheme of in vitro integration. Sequences of HIV-1 proviral 5′- and 3′-ends are shown. Grey segment in the circular plasmid represents the target 144-bp DNA, and the black line represents the remainder of the circular plasmid DNA used for ligation. Red letters in the HIV-1 DNA sequence represent the dinucleotides that were removed in the course of integration. Following incubation of the proviral LTR sequence DNAs with integrase, the resultant re-integrase complexes were reacted with the substrate DNA. PCR amplification was performed using primers in the proviral ends and circular DNA in the substrate DNA and the integration sites were analyzed by direct sequencing. The arrowhead in the PCR product indicates the junction between the provirus and target DNA.
Figure 2.
In vitro integration efficiency and selectivity.
(A) Copy number of PCR products with using primers for the HIV-1 LTR and substrate DNA following integration into the target sequence or random sequences in the substrate DNA, referred to as post-integration amplified products (PIAP). The vertical axis represents log PIAP. Error bars indicate standard deviation (S.E.) (*P<0.001). Plasmid DNA lacking the target sequence was used as a control. (B) Percentages of PIAP copies from the integration into the target sequence or random sequences vs. the total number of PIAP. Dotted line shows the ratio when integration was thought to occur uniformly in the 4-kb substrate DNA. Error bars represent standard deviation (S.E.) (**P<0.001). (C) Number of PIAP copies from integrations into individual segments in x6y6. Vertical axis indicates log PIAP copy number. Error bars represent standard deviation (S.E.) (***P<0.001, 6x&1y vs. other segments).
Figure 3.
Presence of CA and TG motifs and integration ratio.
(A) Number and (B) percentage of PIAP copies from integration into target sequences or sequences lacking 5′-CA-3′ or 5′-GT-3′ dinucleotide motifs (*p<0.001; target vs. CA-; **p<0.001 vs. GT- ; ***p<0.001 vs. CA-GT-; **** p<0.001; target vs. CA-, GT-, CA-GT-). Error bars represent standard deviation (S.E.). The sequences shown display the 6x1y segment of the target DNA. The sequence CA-TG- in Figure 1 indicates an example of CA-TG- sequences.
Figure 4.
In vitro integration using modified or variable-length target sequences.
Number of PIAP copies from integration into variable-length repeat sequences (left). Line represents a linear regression between the logarithm of copy number and number of repeated x, y units. The correlation coefficient was equivalent to 0.96. Repeat number 0 indicates that plasmid DNA was used as the substrate. Error bars represent standard deviation (S.E.). Percentage of PIAP copies from integration into the variable repeat sequences against that from integration into the whole sequence (right). Line represents linear regression between the percentage and number of repeated x, y units. The correlation coefficient was equivalent to 0.92. Error bars represent standard deviation (S.E.) (*P<0.001).
Figure 5.
Interference effects of coexisting modified DNA sequences.
(A)Number and percentage of PIAP copies from integration into the target sequence or into modified sequences I or II. Control was plasmid DNA. Error bars represent standard deviation (S.E.) (*P<0.001). (B)(C) Individual bars show logarithms of number of PIAP copies (B) and percentage of PIAP copies (C) using substrate DNA including the target sequence alone, target plus modified sequence I (left), or target plus modified sequence II (right). The amounts of target and modified sequences were equivalent. Plasmid DNA was used as a control. The percentage was calculated from the ratio of PIAP copies from integration into the target sequence against that from integration into the whole substrate DNA (*, **P<0.05). Error bars represent standard deviation (S.E.).
Figure 6.
Concentration of MnCl2 and integration efficiency/selectivity.
(A) The photo shows electrophoresis of digested substrate DNA in buffer containing 20 mM or 40 mM MnCl2 following incubation for 0, 50 and 90 minutes. An arrow indicates the digested 4.0-kb linearized DNA. (B) Relative area intensity of digested 4.0-kb substrate DNA after electrophoresis. Error bars represent standard deviation (S.E.). The graph shows the area ratio of the digested DNA band on electrophoresis in (A). Error bars represent standard deviation (S.E.). (C) Number of PIAP copies in buffer containing various concentrations of MnCl2. Error bars represent standard deviation (S.E.). (D) Percentage of PIAP copies in buffer containing various concentrations of MnCl2. Error bars represent standard deviation (S.E.).