The effect of Me2+ cofactors at the initial stages of V(D)J recombination.
V(D)J site-specific recombination mediates the somatic assembly of the antigen receptor gene segments. This process is initiated by the recombination activating proteins RAG1 and RAG2, which recognize the recombination signal sequences (RSS) and cleave the DNA at the coding/RSS junction. In this study, we show that RAG1 and RAG2 have the ability to directly interact in solution before binding to the DNA. RAG1 forms a homodimer, which leads to the appearance of two distinct RAG1.RAG2 complexes bound to DNA. To investigate the properties of the two RAG1.RAG2 complexes in the presence of different Me2+ cofactors, we established an in vitro Mg2+-based cleavage reaction on a single RSS. Using this system, we found that Mg2+ confers a specific pattern of DNA binding and cleavage. In contrast, Mn2+ allows aberrant binding of RAG1.RAG2 to single-stranded RSS and permits cleavage independent of binding to the nonamer. To determine the contribution of Me2+ ions at the early stages of V(D)J recombination, we analyzed specific DNA recognition and cleavage by RAG1.RAG2 on phosphorothioated substrates. These experiments revealed that Me2+ ions directly coordinate the binding of RAG1.RAG2 to the RSS DNA.
Santagata
S
S
Mount Sinai School of Medicine, Howard Hughes Medical Institute, Ruttenberg Cancer Center, New York, New York 10029, USA.
Aidinis
V
V
Spanopoulou
E
E
D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015321:Gene Rearrangement; D016654:Genes, RAG-1; D018398:Homeodomain Proteins / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008969:Molecular Sequence Data
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C064655:V(D)J recombination activating protein 2; C064658:RAG-1 protein; D008345:Manganese; D004247:DNA; D008274:Magnesium