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Real-time monitoring of RAG-catalyzed DNA cleavage unveils dynamic changes in coding end association with the coding end complex.

Wang G, Dhar K, Swanson PC, Levitus M, Chang Y - Nucleic Acids Res. (2012)

Bottom Line: By examining the dynamic fluorescence changes during the cleavage reaction, we compared the stability of CEC assembled with core RAG1 paired with full-length RAG2, core RAG2 or a frameshift RAG2 mutant that was speculated to destabilize the PCC, leading to increased aberrant joining.Interestingly, the RAG2 mutant appears to modulate the structure of the RAG-12RSS pre-cleavage complex.Thus, the fluorescence-based detection offers a sensitive, quantitative and continuous assessment of pre-cleavage complex assembly and CEC stability.

View Article: PubMed Central - PubMed

Affiliation: Center of Infectious Disease and Vaccinology, The Biodesign Institute, School of Life Sciences, Arizona State University, Tempe, AZ 85287, USA.

ABSTRACT
During V(D)J recombination, the RAG1/2 recombinase is thought to play an active role in transferring newly excised recombination ends from the RAG post-cleavage complex (PCC) to the non-homologous end joining (NHEJ) machinery to promote appropriate antigen receptor gene assembly. However, this transfer mechanism is poorly understood, partly because of the technical difficulty in revealing weak association of coding ends (CEs) with one of the PCCs, coding end complex (CEC). Using fluorescence resonance energy transfer (FRET) and anisotropy measurement, we present here real-time monitoring of the RAG1/2-catalyzed cleavage reaction, and provide unequivocal evidence that CEs are retained within the CEC in the presence of Mg(2+). By examining the dynamic fluorescence changes during the cleavage reaction, we compared the stability of CEC assembled with core RAG1 paired with full-length RAG2, core RAG2 or a frameshift RAG2 mutant that was speculated to destabilize the PCC, leading to increased aberrant joining. While the latter two CECs exhibit similar stability, the full-length RAG2 renders a less stable CEC unless H3K4me3 peptides are added. Interestingly, the RAG2 mutant appears to modulate the structure of the RAG-12RSS pre-cleavage complex. Thus, the fluorescence-based detection offers a sensitive, quantitative and continuous assessment of pre-cleavage complex assembly and CEC stability.

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The influence of HP-CE release by different cations. (A) FRET changes over the course of cation addition. The reaction was initiated with e/cRAG in Mg2+ and incubated for 2 h, after which Mg2+ (black), Ca2+ (magenta) or Mn2+ (blue) were added. A mock treatment is shown in cyan. (B). Denaturing gel electrophroresis analysis of HP-CE production after the step-wise addition of different cations. The different bands (from top to bottom) represent the intact RSS, hairpin and nicked DNA. All reactions were initiated by e/cRAG in Mg2+ and incubated for 2 h, after which the different cations described in the figure were added to the reaction. The times represent the incubation time after cation addition.
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gks255-F3: The influence of HP-CE release by different cations. (A) FRET changes over the course of cation addition. The reaction was initiated with e/cRAG in Mg2+ and incubated for 2 h, after which Mg2+ (black), Ca2+ (magenta) or Mn2+ (blue) were added. A mock treatment is shown in cyan. (B). Denaturing gel electrophroresis analysis of HP-CE production after the step-wise addition of different cations. The different bands (from top to bottom) represent the intact RSS, hairpin and nicked DNA. All reactions were initiated by e/cRAG in Mg2+ and incubated for 2 h, after which the different cations described in the figure were added to the reaction. The times represent the incubation time after cation addition.

Mentions: To alleviate the complication of bi-phasic FRET decay observed in the e/cRAG/Mn2+ reaction, we conducted the cleavage reaction with step-wise addition of cations. Specifically, we incubated e/cRAG and 12RSS substrate with Mg2+ for 2 h, a time long enough for a sufficient amount of HP-CEs to be generated (Figure 2C), and then added Mg2+, Mn2+ or Ca2+ to focus on the influence of these cations in CEC-SR stability. A mock addition was also included as a control for possible artifacts arising from the pipetting procedure. As shown in Figure 3A, the addition of Mg2+ or Ca2+ resulted in a FRET decrease similar to that obtained with the mock-treatment. However, the addition of Mn2+ resulted in a significant reduction in FRET, causing the dissociation of additional HP-CEs that would otherwise be bound to the protein in the presence of Mg2+. It is important to note that adding Mn2+ after the 2-h incubation in Mg2+ did not result in any further hairpin generation, nor the generation of the unknown band seen in e/cRAG/Mn2+ (compare Figure 2A with Figure 3B), demonstrating that the reduction in FRET is due to hairpin release from the destabilized CEC-SR induced by Mn2+. These results substantiate our notion that the CEC-SR formed in Mg2+ is more stable than the one in Mn2+, and that adding Mn2+ disrupts this complex and causes the premature release of HP-CEs.Figure 3.


Real-time monitoring of RAG-catalyzed DNA cleavage unveils dynamic changes in coding end association with the coding end complex.

Wang G, Dhar K, Swanson PC, Levitus M, Chang Y - Nucleic Acids Res. (2012)

The influence of HP-CE release by different cations. (A) FRET changes over the course of cation addition. The reaction was initiated with e/cRAG in Mg2+ and incubated for 2 h, after which Mg2+ (black), Ca2+ (magenta) or Mn2+ (blue) were added. A mock treatment is shown in cyan. (B). Denaturing gel electrophroresis analysis of HP-CE production after the step-wise addition of different cations. The different bands (from top to bottom) represent the intact RSS, hairpin and nicked DNA. All reactions were initiated by e/cRAG in Mg2+ and incubated for 2 h, after which the different cations described in the figure were added to the reaction. The times represent the incubation time after cation addition.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3401440&req=5

gks255-F3: The influence of HP-CE release by different cations. (A) FRET changes over the course of cation addition. The reaction was initiated with e/cRAG in Mg2+ and incubated for 2 h, after which Mg2+ (black), Ca2+ (magenta) or Mn2+ (blue) were added. A mock treatment is shown in cyan. (B). Denaturing gel electrophroresis analysis of HP-CE production after the step-wise addition of different cations. The different bands (from top to bottom) represent the intact RSS, hairpin and nicked DNA. All reactions were initiated by e/cRAG in Mg2+ and incubated for 2 h, after which the different cations described in the figure were added to the reaction. The times represent the incubation time after cation addition.
Mentions: To alleviate the complication of bi-phasic FRET decay observed in the e/cRAG/Mn2+ reaction, we conducted the cleavage reaction with step-wise addition of cations. Specifically, we incubated e/cRAG and 12RSS substrate with Mg2+ for 2 h, a time long enough for a sufficient amount of HP-CEs to be generated (Figure 2C), and then added Mg2+, Mn2+ or Ca2+ to focus on the influence of these cations in CEC-SR stability. A mock addition was also included as a control for possible artifacts arising from the pipetting procedure. As shown in Figure 3A, the addition of Mg2+ or Ca2+ resulted in a FRET decrease similar to that obtained with the mock-treatment. However, the addition of Mn2+ resulted in a significant reduction in FRET, causing the dissociation of additional HP-CEs that would otherwise be bound to the protein in the presence of Mg2+. It is important to note that adding Mn2+ after the 2-h incubation in Mg2+ did not result in any further hairpin generation, nor the generation of the unknown band seen in e/cRAG/Mn2+ (compare Figure 2A with Figure 3B), demonstrating that the reduction in FRET is due to hairpin release from the destabilized CEC-SR induced by Mn2+. These results substantiate our notion that the CEC-SR formed in Mg2+ is more stable than the one in Mn2+, and that adding Mn2+ disrupts this complex and causes the premature release of HP-CEs.Figure 3.

Bottom Line: By examining the dynamic fluorescence changes during the cleavage reaction, we compared the stability of CEC assembled with core RAG1 paired with full-length RAG2, core RAG2 or a frameshift RAG2 mutant that was speculated to destabilize the PCC, leading to increased aberrant joining.Interestingly, the RAG2 mutant appears to modulate the structure of the RAG-12RSS pre-cleavage complex.Thus, the fluorescence-based detection offers a sensitive, quantitative and continuous assessment of pre-cleavage complex assembly and CEC stability.

View Article: PubMed Central - PubMed

Affiliation: Center of Infectious Disease and Vaccinology, The Biodesign Institute, School of Life Sciences, Arizona State University, Tempe, AZ 85287, USA.

ABSTRACT
During V(D)J recombination, the RAG1/2 recombinase is thought to play an active role in transferring newly excised recombination ends from the RAG post-cleavage complex (PCC) to the non-homologous end joining (NHEJ) machinery to promote appropriate antigen receptor gene assembly. However, this transfer mechanism is poorly understood, partly because of the technical difficulty in revealing weak association of coding ends (CEs) with one of the PCCs, coding end complex (CEC). Using fluorescence resonance energy transfer (FRET) and anisotropy measurement, we present here real-time monitoring of the RAG1/2-catalyzed cleavage reaction, and provide unequivocal evidence that CEs are retained within the CEC in the presence of Mg(2+). By examining the dynamic fluorescence changes during the cleavage reaction, we compared the stability of CEC assembled with core RAG1 paired with full-length RAG2, core RAG2 or a frameshift RAG2 mutant that was speculated to destabilize the PCC, leading to increased aberrant joining. While the latter two CECs exhibit similar stability, the full-length RAG2 renders a less stable CEC unless H3K4me3 peptides are added. Interestingly, the RAG2 mutant appears to modulate the structure of the RAG-12RSS pre-cleavage complex. Thus, the fluorescence-based detection offers a sensitive, quantitative and continuous assessment of pre-cleavage complex assembly and CEC stability.

Show MeSH