Analysis of human immunoglobulin VDJ and DJ rearrangements shows N region synthesis by concatenation of cytosine-rich strands preferentially originating from trimmed germline gene segments

The formation of non-templated (N) regions during immunoglobulin gene rearrangement is a major contributor to antibody diversity. To gain insights into the mechanisms behind this, we studied the nucleotide composition of N regions within 29,962 unique human VHDJH-rearrangements and 8,728 unique human DJH-rearrangements containing exactly one identifiable D-gene segment and thus two N regions, N1 and N2. We found a distinct decreasing content of cytosine (C) and increasing content of guanine (G) across each N region, suggesting that N regions are typically generated by concatenation of two 3-overhangs synthesized by addition of nucleoside triphosphates with a preference for dCTP. This challenges the general assumption that the terminal deoxynucleotidyl transferase favors dGTP in vivo. Furthermore, we found that the G and C gradients depended strongly on whether the germline gene segments were trimmed or not. Our data show that C-enriched N addition preferentially happens at trimmed 3-ends of VH-, D-, and JH-gene segments indicating a dependency of the transferase mechanism upon the nuclease mechanism.