Effect of HIV integrase inhibitors on the RAG1/2 recombinase

Abstract

Assembly of functional Ig and T cell receptor genes by V(D)J recombination depends on site-specific cleavage of chromosomal DNA by the RAG1/2 recombinase. As RAG1/2 action has mechanistic similarities to DNA transposases and integrases such as HIV-1 integrase, we sought to determine how integrase inhibitors of the diketo acid type would affect the various activities of RAG1/2. Both of the inhibitors we tested interfered with DNA cleavage and disintegration activities of RAG1/2, apparently by disrupting interaction with the DNA motifs bound specifically by the recombinase. The inhibitors did not ablate RAG1/2's transposition activity or capture of nonspecific transpositional target DNA, suggesting this DNA occupies a site on the recombinase different from that used for specific binding. These results further underscore the similarities between RAG1/2 and integrase and suggest that certain integrase inhibitors may have the potential to interfere with aspects of B and T cell development.

Figures

Figure 1

Parallels between the biochemical activities of the RAG1/2 recombinase and HIV-1 integrase. (A) RAG1/2 binds to RSS sequences (black triangle) and catalyzes hydrolysis to introduce a nick between the RSS and flanking coding DNA (Nicking). The 3′ hydroxyl (OH) left on coding DNA by this reaction then attacks the opposite strand, leaving a hairpin at the end of coding DNA (Hairpin Formation) and a free 3′ OH at the end of the blunt RSS. The blunt RSS can attack nonspecific target DNA (thick lines; Transposition). Both hairpin formation and transposition are apparently reversible (Open-and-Shut Joint Formation and Disintegration). (B) HIV-1 integrase binds to the long terminal repeats (LTR; gray semicircle) near the ends of viral cDNA. Nicking leaves a free 3′ OH at the end of the LTR (Nicking), which can then attack target DNA (Strand Transfer). Strand transfer is reversible (Disintegration).

Figure 2

Effect of HIV integrase inhibitors on RAG1/2 biochemical activities. The effect of p10 (A) and p8 (B) on nicking and transesterification (hairpin formation) was measured in oligonucleotide cleavage assays performed as described in Materials and Methods. Reaction mixtures were incubated for 30 min after the addition of Mg2+. (C) The effect of p10 on cleavage and disintegration was determined as described in Materials and Methods. All reactions included 10% DMSO. In all cases, inhibitor was added before divalent metal cation. Percent inhibition relative to solvent control is shown.

Figure 3

Mobility-shift assays to examine the effect of p10 on RAG1/2 interaction with RSS. (A) Scheme for binding experiments. Labeled 12 RSS (2 nM; position of label is indicated by an asterisk), 23 RSS (12.5 nM), RAG1/2 (450 ng), and HMG1 (50 ng) were combined in reaction buffer (5 min, 37°C); Ca2+ was added to 4 mM (5 min, 37°C); Mg2+ was added to 4 mM (5 min, 37°C); glycerol was added to 10%, and reactions were loaded onto shift gels. 100% DMSO (1 μl) or 5 mM p10 in 100% DMSO (1 μl) was added at one of the times indicated (1, 2, or 3). (B) Mobility-shift assays were performed as described with p10 or solvent being added as per condition 1, 2, or 3. Filled arrow, free substrate; empty arrow, complex of RAG1/2 with 12 and 23 RSSs.

Figure 4

Effect of p10 on cleavage competence of RAG1/2–RSS complexes. Reactions were assembled as described in Fig. 3 except that the final incubation was extended to 30 min, at which point reactions were stopped by the addition of formamide to 50%. Products were separated on denaturing polyacrylamide gels, and percent of substrate converted to nicked product (A) and hairpin product (B) was determined. “Pre,” “Ca,” and “Mg” correspond to conditions 1, 2, and 3 in Fig. 3; reactions included 10% DMSO (−p10) or 10% DMSO plus 0.5 mM p10 (+p10). Results are the average of three independent trials with standard deviation indicated by error bars.

Figure 5

Effect of p10 on target capture and transposition. (A) Unlabeled 12 and 23 RSSs (50 nM each), RAG1/2 (450 ng), and HMG1 (50 ng) were combined in reaction buffer (5 min, 37°C); Ca2+ was added to 4 mM (5 min, 37°C); Mg2+ was added to 4 mM (30 min, 37°C); glycerol was added to 10%, and reactions were loaded onto shift gels. Reaction volume (1/10) of 100% DMSO or 5 mM p10 in 100% DMSO and labeled target oligonucleotide (10 nM) were added under conditions 1 (Pre), 2 (Ca), or 3 (Mg), as described in Fig. 3. Activity as percent of solvent control is shown. Open arrow, target capture complex. (B) Labeled 12 RSS and unlabeled 23 RSS (50 nM each), RAG1/2 (450 ng), and HMG1 (50 ng) were combined in reaction buffer (5 min, 37°C); Ca2+ was added to 4 mM (5 min, 37°C); Mg2+ was added to 4 mM (60 min, 37°C). p10 was then added to the concentration indicated, followed by the addition of 100 ng of supercoiled pBR322 target DNA (30 min, 37°C). Deproteinized products were separated on agarose gels. Open arrow, single-ended transposition product; filled arrow, double-ended transposition product. Activity as percent of solvent control is shown.