Structure‐specific nuclease activity of RAGs is modulated by sequence, length and phase position of flanking double‐stranded DNA

Abstract

RAGs (recombination activating genes) are responsible for the generation of antigen receptor diversity through the process of combinatorial joining of different V (variable), D (diversity) and J (joining) gene segments. In addition to its physiological property, wherein RAG functions as a sequence-specific nuclease, it can also act as a structure-specific nuclease leading to genomic instability and cancer. In the present study, we investigate the factors that regulate RAG cleavage on non-B DNA structures. We find that RAG binding and cleavage on heteroduplex DNA is dependent on the length of the double-stranded flanking region. Besides, the immediate flanking doublestranded region regulates RAG activity in a sequence-dependent manner. Interestingly, the cleavage efficiency of RAGs at the heteroduplex region is influenced by the phasing of DNA. Thus, our results suggest that sequence, length and phase positions of the DNA can affect the efficiency of RAG cleavage when it acts as a structure-specific nuclease. These findings provide novel insights on the regulation of the pathological functions of RAGs.

HIV integrase inhibitor, Elvitegravir, impairs RAG functions and inhibits V(D)J recombination

“…41 The protein was purified as described previously. 53, 54, 55 In brief, HEK 293 T cells were transfected with plasmid constructs using Calcium phosphate method. Cells were harvested after 48 h of transfection, protein lysate was prepared and RAG proteins were purified using amylose resin column chromatography.…”

“…53, 54, 57 In brief, appropriate [γ- 32 P] ATP-labelled oligomeric DNA substrates were incubated with RAG proteins (100 ng) in a buffer containing 22.5 mM MOPS-KOH (pH 7.0), 20% DMSO, 2.2 mM DTT, 50 mM potassium glutamate, 2% (v/v) glycerol and 100 ng/ml BSA for 2 h at 25 °C following which fixation was carried out (0.01% v/v gluteraldehyde at 37 °C for 10 min). The DNA–protein complexes were allowed to form in the presence of 5 mM CaCl 2 and increasing concentration of integrase inhibitors (0, 0.1, 0.2, 0.3 and 0.5 mM) and resolved on native polyacrylamide gels (6%).…”

Identification and characterization of novel SCR7‐based small‐molecule inhibitor of DNA end‐joining, SCR130 and its relevance in cancer therapeutics

“…Oligomeric DNA, obtained from Macrogen (Seoul, South Korea) was purified as described earlier. 27 Refer the Supporting Information for a sequence of oligomers used in the study.…”

Snaps and mends: DNA breaks and chromosomal translocations

“…). However, recent studies showed that the sequence of both the single‐stranded region of the non‐B‐DNA and its flanking DNA sequences may regulate RAG cleavage at such structures . On cleavage, signal joints are formed that may be insert at a target site due to the transposase activity of RAGs .…”