MECHANISTIC CONSTRAINTS ON DIVERSITY IN HUMAN V(D)J RECOMBINATION

We have analyzed a large collection of coding junctions generated in h uman cells. From this analysis, we infer the following about nucleotid e processing at coding joints in human cells, First, the pattern of nu cleotide loss from coding ends is influenced by the base composition o f the coding end sequences. AT-rich sequences suffer greater loss than do GC-rich sequences, Second, inverted repents can occur at ends that have undergone nucleolytic processing, Previously, inverted repents ( P nucleotides) have been noted only at coding ends that have not under gone nucleolytic processing, this observation being the basis for a mo del 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 nucleotid e additions, Terminal deoxynucleotidyl transferase (TdT) is required f or the appearance of the inverted repeats at processed ends (but not f ull-length coding ends), get statistical analysis shows that it is vir tually impossible for the inverted repeats to be polymerized by TdT, T hird, TdT additions are not random, It has long been noted that TdT ha s a G utilization preference. In addition to the G preference, we find that TdT adds strings of purines or strings of pyrimidines at a highl y significant frequency. This tendency suggests that nucleotide-stacki ng interactions affect TdT polymerization. All three of these features place constraints on the extent of junctional diversity in human V(D) J recombination.