V(D)J recombination activity in human hematopoietic cells: correlation with developmental stage and genome stability.

V(D)J recombinase activity was measured in an array of human cell lines derived from hematopoietic malignancies representing various lineages and developmental stages. The level of recombinase activity was found to vary over a 2000-fold range between different cell lines. Several myeloid cell lines were positive for V(D)J recombinase activity, providing additional insight into the relationship between myeloid and lymphoid differentiation. Despite high levels of V(D)J recombination in two human acute lymphoblastic leukemia cell lines, the cytogenetic karyotype has remained essentially constant over several years of continuous cell culture. Silencing of recombination of chromosomal and minichromosomal targets has been strongly correlated with the replication of CpG methylated DNA in murine cells. Here, in human cells, we show that human minichromosomes bearing V(D)J recombination signals are protected well over 100-fold from recombination if they are CpG methylated, providing a rational basis for the karyotypic stability in cells with high levels of V(D)J recombination activity.

Major domain swiveling revealed by the crystal structures of complexes of E. coli Rep helicase bound to single-stranded DNA and ADP.

Crystal structures of binary and ternary complexes of the E. coli Rep helicase bound to single-stranded (ss) DNA or ssDNA and ADP were determined to a resolution of 3.0 A and 3.2 A, respectively. The asymmetric unit in the crystals contains two Rep monomers differing from each other by a large reorientation of one of the domains, corresponding to a swiveling of 130 degrees about a hinge region. Such domain movements are sufficiently large to suggest that these may be coupled to translocation of the Rep dimer along DNA. The ssDNA binding site involves the helicase motifs Ia, III, and V, whereas the ADP binding site involves helicase motifs I and IV. Residues in motifs II and VI may function to transduce the allosteric effects of nucleotides on DNA binding. These structures represent the first view of a DNA helicase bound to DNA.

RAG mutations in human B cell-negative SCID.

Patients with human severe combined immunodeficiency (SCID) can be divided into those with B lymphocytes (B+ SCID) and those without (B- SCID). Although several genetic causes are known for B+ SCID, the etiology of B- SCID has not been defined. Six of 14 B- SCID patients tested were found to carry a mutation of the recombinase activating gene 1 (RAG-1), RAG-2, or both. This mutation resulted in a functional inability to form antigen receptors through genetic recombination and links a defect in one of the site-specific recombination systems to a human disease.

Mechanistic constraints on diversity in human V(D)J recombination.

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.

Unequal signal and coding joint formation in human V(D)J recombination.

Substrates for studying V(D)J recombination in human cells and two human pre-B-cell lines that have active V(D)J recombination activity are described. Using these substrates, we have been able to analyze the relative efficiency of signal joint and coding joint formation. Coding joint formation was five- to sixfold less efficient than signal joint formation in both cell lines. This imbalance between the two halves of the reaction was demonstrated on deletional substrates, where each joint is assayed individually. In both cell lines, the inversional reaction (which requires formation of both a signal and a coding joint) was more than 20-fold less efficient than signal joint formation alone. The signal and coding sequences are identical in all of these substrates. Hence, the basis for these differential reaction ratios appears to be that coding joint and signal joint formation are both inefficient and their combined effects are such that inversions (two-joint reactions) reflect the product of these inefficiencies. Physiologically, these results have two implications. First, they show how signal and coding joint formation efficiencies can affect the ratio of deletional to inversional products at endogenous loci. Second, the fact that not all signal and coding joints go to completion implies that the recombinase is generating numerous broken ends. Such unresolved ends may participate in pathologic chromosomal rearrangements even when the other half of the same reaction may have proceeded to resolution.

The basis for the mechanistic bias for deletional over inversional V(D)J recombination.

V(D)J recombination between recognition sites in the genome is characterized by certain biases. At some loci, proximal sites undergo recombination substantially more frequently than distal ones. The joining of DH/JH is an example of this. Because the DH element bears signal sequences on each side, inversion would be expected as often as deletion in DH/JH recombination. However, the markedly favored outcome is deletion, entailing utilization of the closer recombination site. One model proposed to explain these biases is the tracking model in which the recombinase tracks from one site to the other. Here, we have directly tested for various types of tracking in V(D)J recombination and have found no indication that it occurs. In addition, we have created DH-JH minilocus substrates for analysis of the basis for the preference for deletion. We find that we can reproduce the deletional bias for the system. Moreover, by flipping the orientation of the D segment, we can reverse the bias such that the frequency of inversions can exceed the number of deletions. These results indicate (1) that there is no intrinsic topological preference in this reaction, and (2) that the sequence of the signal and coding ends determines the bias.