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 double-strand 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.
Highlights
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
We previously showed that a non-B DNA structure formed at the BCL2 major breakpoint region (MBR) on chromosome 18 involved in t(14;18) translocation in follicular lymphoma can be cleaved by RAGs [25,26,27,28,29]
Preliminary results showed that the efficiency of RAG cleavage on heteroduplex DNA is dependent on the sequence composition of the bubble
Summary
Chemicals, and Reagents—Chemical reagents were obtained from Sigma, Amresco, and SRL. RAG Cleavage on Oligomeric DNA—Appropriate oligomeric substrates were incubated with RAG proteins for 1 h at 37 °C in a buffer containing 25 mM MOPS, (pH 7.0), 30 mM KCl, 30 mM potassium glutamate, and 5 mM MgCl2 as described earlier [30, 31]. P1 Nuclease Cleavage Assay—The substrate DNA containing different types of bubble sequences was incubated with P1 nuclease as described earlier [41]. A 5Ј endlabeled substrate DNA was tested for P1 nuclease sensitivity by incubating with increasing concentrations (0.001, 0.01, and 0.1 units) of P1 nuclease in a buffer containing 10 mM Tris-HCl (pH 7.9), 10 mM MgCl2 ,50 mM NaCl, and 1 mM DTT at 37 °C for 30 min. Reaction products were resolved on a 12–15% denaturing PAGE and analyzed
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