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Summary Long DNA palindromes are implicated in chromosomal rearrangement, but their roles in the underlying molecular events remain a matter of conjecture. One notion is that palindromes induce DNA breaks after assuming a cruciform structure, the four-way DNA junction providing a target for cleavage by Holliday junction (HJ)-specific enzymes. Though compelling, few components of the "cruciform resolution" proposal are established. Here we address fundamental properties and genetic dependencies of palindromic DNA metabolism in eukaryotes. Plasmid-borne palindromes introduced into S. cerevisiae are site-specifically broken in vivo, and the breaks exhibit unique hallmarks of an HJ resolvase mechanism. In vivo resolution requires Mus81, for which the bacterial HJ resolvase RusA will substitute. These results provide confirmation of cruciform extrusion and resolution in the context of eukaryotic chromatin. Related observations are that, unchecked by a nuclease function provided by Mre11, episomal palindromes launch a self-perpetuating breakage-fusion-bridge-independent copy number increase termed "escape."

In mice, kappa light chains dominate over lambda in the immunoglobulin repertoire by as much as 20-fold. Although a major contributor to this difference is the recombination signal sequences (RSS), the mechanism by which RSS cause differential representation has not been determined. To elucidate the mechanism, we tested kappa and lambda RSS flanked by their natural 5' and 3' flanks in three systems that monitor V(D)J recombination. Using extra-chromosomal recombination substrates, we established that a kappa RSS and its flanks support six- to nine-fold higher levels of recombination than a lambda counterpart. In vitro cleavage assays with these same sequences demonstrated that single cleavage at individual kappa or lambda RSS (plus flanks) occurs with comparable frequencies, but that a pair of kappa RSS (plus flanks) support significantly higher levels of double cleavage than a pair of lambda RSS (plus flanks). Using EMSA with double stranded oligonucleotides containing the same kappa or lambda RSS and their respective flanks, we examined RAG/DNA complex formation. We report that, surprisingly, RAG-1/2 form only modestly higher levels of complexes on individual 12 and 23 kappa RSS (plus natural flanks) as compared to their lambda counterparts. We conclude that the overuse of kappa compared to lambda segments cannot be accounted for by differences in RAG-1/2 binding nor by cleavage at individual RSS but rather could be accounted for by enhanced pair-wise cleavage of kappa RSS by RAG-1/2. Based on the data presented, we suggest that the biased usage of light chain segments is imposed at the level of synaptic RSS pairs.

The fine-structures of V(D)J joining products present a cache of information about the recombination process. Testable hypotheses with predictive value are beginning to provide a surprisingly detailed picture of the joining operation. Here, one feature of V(D)J junctions, the appearance of short palindromic inserts, is discussed, and experimental evidence in support of a joining model involving a hairpin-terminated DNA intermediate will be summarized.

SummaryWhereas chromosomal translocations are common pathogenetic events in cancer, mechanisms that promote them are poorly understood. To elucidate translocation mechanisms in mammalian cells, we developed high-throughput, genome-wide translocation sequencing (HTGTS). We employed HTGTS to identify tens of thousands of independent translocation junctions involving fixed I-SceI meganuclease-generated DNA double-strand breaks (DSBs) within the c-myc oncogene or IgH locus of B lymphocytes induced for activation-induced cytidine deaminase (AID)-dependent IgH class switching. DSBs translocated widely across the genome but were preferentially targeted to transcribed chromosomal regions. Additionally, numerous AID-dependent and AID-independent hot spots were targeted, with the latter comprising mainly cryptic I-SceI targets. Comparison of translocation junctions with genome-wide nuclear run-ons revealed a marked association between transcription start sites and translocation targeting. The majority of translocation junctions were formed via end-joining with short microhomologies. Our findings have implications for diverse fields, including gene therapy and cancer genomics.

We describe novel products of V(D)J recombination in which signal sequences become joined to coding elements, in contrast to the standard reaction whose products are junctions of two signal sequences or two coding elements. In this variant reaction, the recombination machinery evidently recognizes signal sequences and introduces strand breaks at the normal positions, but then connects the elements in unusual combinations. The lack of fixed directionality indicates that recombination sites are not uniquely aligned when strand exchange occurs. The discovery of these variant junctions suggests a model for the evolution of the antigen receptor loci.

A cell line transformed by Abelson murine leukemia virus, called PD, is capable of carrying out kappa-gene rearrangement while growing in culture. Subclones of PD have diverse kappa-gene structures, and some derivatives show evidence of continued joining activity after as many as three subclonings. Analysis of PD sublineages has shown that a rearranged chromosome can undergo secondary kappa-gene rearrangements, producing either a new rearrangement or a deletion of C kappa. Although the PD line actively rearranges its kappa genes, its rearranged heavy-chain genes show little variation, and there is no rearrangement of lambda genes. In PD subclones, DNA fragments representing the reciprocal product of kappa-gene rearrangement are often evident, and they may undergo either further rearrangement or deletion. The implications of multiple rearrangements on a single chromosome and of the maintenance of reciprocal fragments are considered in the context of a model that postulates that the V kappa and J kappa segments are not all organized in the DNA in the same transcriptional direction, leading to inversions rather than deletions during joining.

The structure of immunoglobulin-related gene was analyzed in individual Abelson murine leukemia virus (A-MuLV)-transformed lymphoid cell lines. Essentially all of these lines, whether immunoglobulin-containing or null, had DNA rearrangements in the vicinity of the JH regions on both chromosomes as well as deletions of at least 5 kb of DNA 5' to JH. None of these lines, however, except rare light chain producers, had detectable rearrangement at either their kappa or lambda light chain loci. In contrast to A-MuLV-transformants derived from bone marrow. Those from early fetal liver frequently contained more than two and sometimes 12 or more distinct, rearranged JH-containing fragments. Cellular subclones derived from these lines had a subset, usually two, of the fragments found in the parent line. Therefore, heavy chain gene rearrangement appears to precede that of light chain gene rearrangement and is still continuing in certain cultured A-MuLV transformants.