Fig. 5.11 The introduction of Pand N-nucleotides diversifies the joints between gene segments during immunoglobulin gene rearrangement. The process is illustrated for a DH to JH rearrangement (first panel); however, the same steps occur in VH to DH and in VL to JL rearrangements. After formation of the DNA hairpins (second panel), the two heptamer sequences are ligated to form the signal joint (not shown here), while the Artemis:DNA-PK complex cleaves the DNA hairpin at a random site (indicated by the arrows) to yield a single-stranded DNA end (third panel). Depending on the site of cleavage, this single-stranded DNA may contain nucleotides that were originally complementary in the double-stranded DNA and which therefore form short DNA palindromes, such as TCGA and ATAT, as indicated by the light blue-shaded box. For example, the sequence GA at the end of the D segment shown is complementary to the preceding sequence TC. Such stretches of nucleotides that originate from the complementary strand are known as P-nucleotides. Where the enzyme terminal deoxynucleotidyl transferase (TdT) is present, nucleotides are added at random to the ends of the single-stranded segments (fourth panel); these nontemplated, or N, nucleotides are indicated by the shaded box. The two single-stranded ends then pair (fifth panel). Exonuclease trimming of unpaired nucleotides (sixth panel) and repair of the coding joint by DNA synthesis and ligation (bottom panel) leaves both Pand N-nucleotides (indicated by light blue shading) in the final coding joint. The randomness of insertion of Pand N-nucleotides makes an individual P–N region virtually unique and a valuable marker for following an individual B-cell clone as it develops, for instance in studies of somatic hypermutation.
TCGCCGGCAGATATATATGCGCATATPPNDJThe gaps are ÿlled by DNA synthesis and ligation to form coding joint TCCGGCAATGCTTAADJUnpaired nucleotides are removed by an exonuclease TCCGGCTAAATGCTTAAAATGCTCCGGCTATTAAATGCTTAADJPairing of strands DJN-nucleotide additions by TdT JDArtemis:DNA-PK complex opens DNA hairpins, generating palindromic P-nucleotides ATATATGCJDRAG complex generates DNA hairpin at coding ends RSSs brought together ATATATGCGCGCATATATGCGCGCGCATATATGCGCJD strand (see Fig. 5.11). In many light-chain gene rearrangements, DNA repair enzymes then fill in complementary nucleotides on the single-stranded tails, which would leave short palindromic sequences (the P-nucleotides) at the joint if the ends were rejoined without any further exonuclease activity.