Cytosines, but Not Purines, Determine Recombination Activating Gene (RAG)-induced Breaks on Heteroduplex DNA Structures

Abstract

The sequence specificity of the recombination activating gene (RAG) complex during V(D)J recombination has been well studied. RAGs can also act as structure-specific nuclease; however, little is known about the mechanism of its action. Here, we show that in addition to DNA structure, sequence dictates the pattern and efficiency of RAG cleavage on altered DNA structures. Cytosine nucleotides are preferentially nicked by RAGs when present at single-stranded regions of heteroduplex DNA. Although unpaired thymine nucleotides are also nicked, the efficiency is many fold weaker. Induction of single-or doublestrand breaks by RAGs depends on the position of cytosines and whether it is present on one or both of the strands. Interestingly, RAGs are unable to induce breaks when adenine or guanine nucleotides are present at single-strand regions. The nucleotide present immediately next to the bubble sequence could also affect RAG cleavage. Hence, we propose "C (d) C (S) C (S) " (d, double-stranded; s, single-stranded) as a consensus sequence for RAG-induced breaks at single-/double-strand DNA transitions. Such a consensus sequence motif is useful for explaining RAG cleavage on other types of DNA structures described in the literature. Therefore, the mechanism of RAG cleavage described here could explain facets of chromosomal rearrangements specific to lymphoid tissues leading to genomic instability. The recombination activating gene (RAG)3 complex, consisting of RAG1 and RAG2, is the nuclease responsible for V(D)J recombination, a physiological process by which immunoglobulin and T-cell receptor diversity is generated. RAGs are normally expressed in B-cells and T-cells (1). During V(D)J recombination, the variable (V), diversity (D), and joining (J) subexons are rearranged. Specific sequences present at the ends of the subexon, called recombination signal sequences (RSS) are recognized by RAGs. Each RSS consists of a conserved heptamer and nonamer, separated by a nonconserved spacer, the length of which designates RSS as a 12-signal or 23-signal. Normally during V(D)J recombination, a 12-signal pairs with a 23-signal with the help of proteins like HMGB1 (high mobility group box 1). The nick induced by RAGs during V(D)J recombination is consistently 5Ј of the heptamers (2-14). The nicked strand is then converted to a hairpin in each V, D, and J coding end by a transesterification reaction, leaving each of the signal ends blunt (15). The hairpins are then opened by the ArtemisDNAPKcs complex (16). After cleavage, the RAG complex remains tightly bound to the two signal ends and less tightly bound to the coding end, in a postcleavage complex (17,18). Finally, the complete exon coding for antibody or TCR (T-cell receptor) is generated by joining of the broken subexons by nonhomologous DNA endjoining (NHEJ) (19,20).In the recent past, studies have shown that cryptic RSS sites present elsewhere in the genome can also act as off-target sites for RAG misrecognition, leading to chromosomal translocations in lymphoid cancers such as l...

Mechanism of Fragility at BCL2 Gene Minor Breakpoint Cluster Region during t(14;18) Chromosomal Translocation

“…RAG Expression and Purification-Human GST and MBP core RAG1 (384 -1008 amino acids; cRAG1) and core RAG2 (1-383 amino acids; cRAG2) proteins were purified as described (35,36). The protein expression was checked by Western blotting by using the appropriate antibodies (Santa Cruz Biotechnology) (supplemental Fig.…”

“…The double-strand break thus generated is a prerequisite for chromosomal translocations. Another RAG-mediated mechanism in translocations utilizes the novel property of RAGs being a structure-specific nuclease (33)(34)(35)(36). It has been shown that a non-B DNA structure formed at BCL2 MBR could be cleaved by RAGs, leading to t(14;18) translocation (3,33,37).…”

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

“…It has been shown that RAGs can cleave different altered DNA structures or end configurations in physiological ionic conditions [11][12][13][14][15][16][17]. Besides, RAGs can also cleave hairpin structures in the presence of Mn 2+ [18,19].…”

“…The presence of cytosine and thymine at the single-stranded (ss) region of heteroduplex DNA is critical for RAG nicking and double-strand break formation [12]. A minimum of two cytosines at the ss region of heteroduplex DNA is required for efficient RAG cleavage [12].…”

“…Apart from the classical function of RAGs, as a sequence-specific nuclease, recent studies have shown that RAGs possess structure-specific nuclease activity [9][10][11][12][13][14]. It has been shown that RAGs can cleave different altered DNA structures or end configurations in physiological ionic conditions [11][12][13][14][15][16][17].…”