Mechanistic Constraints on Diversity in Human V(D)J Recombination

Abstract

We have analyzed a large collection of coding junctions generated in human cells. From this analysis, we infer the following about nucleotide processing at coding joints in human cells. First, the pattern of nucleotide loss from coding ends is influenced by the base composition of the coding end sequences. AT-rich sequences suffer greater loss than do GC-rich sequences. Second, inverted repeats can occur at ends that have undergone nucleolytic processing. Previously, inverted repeats (P nucleotides) have been noted only at coding ends that have not undergone nucleolytic processing, this observation being the basis for a model in which a hairpin intermediate is formed at the coding ends early in the reaction. Here, inverted repeats at processed coding ends were present at approximately twice the number of junctions as P nucleotide additions. Terminal deoxynucleotidyl transferase (TdT) is required for the appearance of the inverted repeats at processed ends (but not full-length coding ends), yet statistical analysis shows that it is virtually impossible for the inverted repeats to be polymerized by TdT. Third, TdT additions are not random. It has long been noted that TdT has a G utilization preference. In addition to the G preference, we find that TdT adds strings of purines or strings of pyrimidines at a highly significant frequency. This tendency suggests that nucleotide-stacking interactions affect TdT polymerization. All three of these features place constraints on the extent of junctional diversity in human V(D)J recombination.

Variable Diversity Joining Recombination: Nonhairpin Coding Ends in Thymocytes of SCID and Wild-Type Mice

“…Assuming that these blunt ends were generated from a hairpin precursor, coding sequence could affect processing by influencing the site of hairpin opening and the extent of subsequent nucleotide loss. This explanation was proposed in studies of coding joints at endogenous loci (49) and extra-chromosomal substrates (50,51) in which nucleotide deletion distinct for different coding segments was noted. Whether the blunt coding ends reported in this study are used for coding joints or represent dead-end intermediates is unclear.…”

Predicting the spectrum of TCR repertoire sharing with a data‐driven model of recombination

“…The germline gene usages are highly non‐uniform,14, 15, 33 due to differences in gene copy numbers34 as well as the conformation35 and processive excision dynamics36 of DNA during recombination. In addition, the distributions of the number of deleted and inserted base pairs, as well as the composition of N nucleotides, are also biased 37. Taken together, the biases imply that some recombination events are more likely than others.…”

The VλJλ Repertoire in Human Fetal Spleen: Evidence for Positive Selection and Extensive Receptor Editing

“…These results imply that the expressed fetal repertoire is both positively and negatively selected based upon junctional diversity. Selection of rearrangements with H joining causes major constraints on the junctional diversity mechanism observed in the productive repertoire, which appears to be the major outcome of selection in the fetus (17,43).…”