Regulating antigen-receptor gene assembly (original) (raw)

2003, Nature Reviews Immunology

RECOMBINATION SIGNAL SEQUENCES (RSSs). Short, conserved DNA sequences that flank all rearranging gene segments and serve as the recognition elements for the recombinase machinery. Heptamer CACAGTG Spacer Nonamer ACAAAAAC Box 1 | The V(D)J recombination pathway: signals, intermediates, products and factors Rearranging gene segments are flanked by recombination signal sequences (RSSs), which consist of a conserved heptamer and nonamer separated by a non-conserved spacer of either 12 or 23 nucleotides (+ or-1 nucleotide) in length. Gene segments of a particular type, variable heavy-chain (V H) gene segments for example, are all flanked by RSSs with the same spacer length (23 nucleotides, in this case). Only gene segments that are flanked by RSSs with dissimilar spacer lengths can recombine with one another due to a limitation known as the 12/23 rule. Recombinase-activating gene 1 (RAG1) and RAG2 form a heteromultimer that recognizes and cleaves a 12/23 pair of RSSs precisely at the heptamer-RSS junction, generating reaction intermediates that consist of four DNA ends. The consensus heptamer and nonamer sequences are shown. High mobility group 1 (HMG1)-a prevalent non-histone chromosomal protein-stimulates this reaction in vitro, but its role in vivo remains undefined. Coding ends are covalently closed DNA hairpins and signal ends are blunt and 5′ phosphorylated. The latter stages of V(D)J recombination require the promiscuously expressed non-homologous end-joining (NHEJ) proteins DNA-dependent protein kinase (DNA-PK), Ku, X-ray repair cross complementing protein 4 (XRCC4), DNA ligase IV and Artemis in addition to the RAG proteins. Signal ends are joined by DNA ligase IV to form a signal joint and coding ends are first opened by the nuclease activity of Artemis, processed, then joined by DNA ligase IV to form a coding joint. Coding joints are imprecise and contain short deletions, palindromic duplications (P-segments) or non-templated nucleotide additions (N-regions) (introduced by terminal deoxynucleotidyl transferase, TDT). Ku70 is a DNA end-binding protein that interacts with Ku80. Ku80 in turn recruits DNA-PK, a protein kinase that is required to activate the nuclease activity of Artemis. XRCC4 increases the activity of DNA ligase IV. Null mutations in any of these proteins blocks the formation of coding joints, whereas mutation of DNA-PK spares signaljoint formation. As the joining step has not been recapitulated in vitro using purified components, it remains possible that additional factors might be required. Similarly, it is possible that specific factors might enhance RSS recognition or cleavage by the RAG proteins in vivo. (See REF. 4 for a recent review of the V(D)J recombination pathway).