Modeling of the RAG reaction mechanism

Abstract

In vertebrate V(D)J recombination, it remains unclear how the RAG complex coordinates its catalytic steps with binding to two distant recombination sites. Here, we test the ability of the plausible reaction schemes to fit observed time courses for RAG nicking and DNA hairpin formation. The reaction schemes with the best fitting capability (1) strongly favor a RAG tetrameric complex over a RAG octameric complex; (2) indicate that once a RAG complex brings two recombination signal sequence (RSS) sites into synapsis, the synaptic complex rarely disassembles; (3) predict that the binding of both RSS sites (synapsis) occurs before catalysis (nicking); and (4) show that the RAG binding properties permit strong distinction between RSS sites within active chromatin versus nonspecific DNA or RSS sites within inactive chromatin. The results provide general insights for synapsis by nuclear proteins as well as more specific testable predictions for the RAG proteins.

Figure 1. Kinetics of coupled cleavage assay with the full-length RAG complex

(A) Time course of nicking at 12RSS, hairpinning at 12RSS, and undigested 12RSS substrate. Data points represent the average and error bars show the range. These experiments were repeated twice, and related time courses indicate that these time courses are quite representative. (B) The table shows numerical values of concentration in nM for 12RSS, nicked 12RSS and hairpin product over the time course of the reaction (see Experimental Procedures for details).

Figure 2. Schematics of the potential mechanisms of RAG nicking and hairpin formation

The RAG catalytic complex can carry out nicking and hairpin formation in at least eight mechanistically distinguishable ways. Reaction schemes 1 to 4 are shown here. For other reaction schemes please see Figure 3. Catalytic rate constants are shown next to the corresponding reaction arrows by lowercase letters with subscripts. See text for details. R, RAG enzymatic complex; 12, 12RSS; 23, 23RSS; R:12, RAG bound to 12RSS; R:23, RAG bound to 23RSS; N, nicked; H, hairpin.

Figure 3. Schematics of the potential mechanisms of RAG nicking and hairpin formation

The RAG catalytic complex can carry out nicking and hairpin formation in at least eight mechanistically distinguishable ways. Reaction schemes 5 to 8 are shown here. Catalytic rate constants are shown next to the corresponding reaction arrows by lowercase letters with subscripts. See text for details. R, RAG enzymatic complex; 12, 12RSS; 23, 23RSS; R:12, RAG bound to 12RSS; R:23, RAG bound to 23RSS; N, nicked; H, hairpin. See also Figure 2.

Figure 4. The outcome of curve fitting for Models 1, 5, 7, and 8

Predicted concentrations of 12RSS substrate (blue), nicked 12RSS (green), and hairpin 12RSS (red) based on the best fit of (A) scheme 1, (B) scheme 5, (C) scheme 7, and (D) scheme 8 are shown in solid lines. Experimentally measured concentrations of 12RSS, nicked 12RSS, and hairpin 12RSS are shown in points with error bars as explained in Figure 1. See text for more information on conditions and assumptions of curve fitting. Schemes 7 and 8 provide similarly good fits for other RAG couple-cleavage time courses that start with different substrate concentrations (data not shown).

Figure 5. Probability of RAG binding at RSS sites in active and inactive chromatin as a function of free RAG concentration

The probability of RAG binding at any position in the genome depends on the presence or absence of H3K4me3 marks as well as RSS sites at that position. The likelihood of a non-specific DNA region, an RSS without H3K4me3 mark, a region with two H3K4me3 marks but without RSS sequence, and an RSS with one, or two H3K4me3 marks to be occupied by RAG complex are shown here as a function of free RAG concentration in the nucleus.