Joanne E. Hesse

The defect in murine severe combined immune deficiency: Joining of signal sequences but not coding segments in V(D)J recombination

Pre-B and pre-T cell lines from mutant mice with severe combined immune deficiency (scid mice) were transfected with plasmids that contained recombination signal sequences of antigen receptor gene elements (V, D, and J). Recovered plasmids were tested for possible recombination of signal sequences and/or the adjacent (coding) sequences. Signal ends were joined, but recombination was abnormal in that half of the recombinants had lost nucleotides from one or both signals. Coding ends were not joined at all in either deletional or inversional V(D)J recombination reactions. However, coding ends were able to participate in alternative reactions. The failure of coding joint formation in scid pre-B and pre-T cells appears sufficient to explain the absence of immunoglobulin or T cell receptor production in scid mice.

Extrachromosomal DNA substrates in pre-B cells undergo inversion or deletion at immunoglobulin V-(D)-J joining signals.

Sequences encoding immunoglobulin variable domains are known to be assembled from variable (V), diversity (D), and joining (J) segments by site-specific recombination. We present a sensitive and rapid assay for V-(D)-J recombination that uses plasmid DNA transiently introduced into transformed pre-B cells, and demonstrates that the recombination is independent of any unique chromosomal context. Sequences sufficient to constitute recombination sites are contained within the 84 and 42 bp flanking, respectively, the murine J kappa 1 and V kappa L8 segments, which include the known heptamer-nonamer V-(D)-J joining signals. Deletion and inversion occur at comparable frequencies. Thus, V-(D)-J recombination may be relatively insensitive to the topological arrangement of sites, and events at the two novel junctions produced by the reaction may be coupled.

DNA binding of Xrcc4 protein is associated with V(D)J recombination but not with stimulation of DNA ligase IV activity

Mammalian cells are protected from the effects of DNA double‐strand breaks by end‐joining repair. Cells lacking the Xrcc4 protein are hypersensitive to agents that induce DNA double‐strand breaks, and are unable to complete V(D)J recombination. The residual repair of broken DNA ends in XRCC4‐deficient cells requires short sequence homologies, thus possibly implicating Xrcc4 in end alignment. We show that Xrcc4 binds DNA, and prefers DNA with nicks or broken ends. Xrcc4 also binds to DNA ligase IV and enhances its joining activity. This stimulatory effect is shown to occur at the adenylation of the enzyme. DNA binding of Xrcc4 is correlated with its complementation of the V(D)J recombination defects in XRCC4‐deficient cells, but is not required for stimulation of DNA ligase IV. Thus, the ability of Xrcc4 to bind to DNA suggests functions independent of DNA ligase IV.

Novel strand exchanges in V(D)J recombination

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.

Initiation of V(D)J Recombination in a Cell-Free System by RAG1 and RAG2 Proteins

Mature T cell receptor (TCR) and immunoglobulin (Ig) genes are assembled from separate gene segments, which are flanked by recombination signal sequences (RSS), consisting of conserved heptamer and nonamer motifs separated by a spacer region of 12 or 23 base pairs (bp). V(D)J recombination results in a precise head-to-head ligation of two signal sequences in a signal joint, and the imprecise joining of two coding segments in a coding joint, which may contain additions or deletions of a few bp. The imprecise nature of coding joints led to the hypothesis that double strand breaks (DSB) might be intermediates in the recombination pathway These DSB can then be processed by an exonuclease and/or by terminal deoxynucleotidyl transferase (TdT) before formation of a coding joint.

Requirements for DNA hairpin formation by RAG1/2

The rearrangement of antigen receptor genes is initiated by double-strand breaks catalyzed by the RAG1/2 complex at the junctions of recombination signal sequences and coding segments. As with some “cut-and-paste” transposases, such as Tn5 and Hermes, a DNA hairpin is formed at one end of the break via a nicked intermediate. By using abasic DNA substrates, we show that different base positions are important for the two steps of cleavage. Removal of one base in the coding flank enhances hairpin formation, bypassing a requirement for a paired complex of two signal sequences. Rescue by abasic substrates is consistent with a base-flip mechanism seen in the crystal structure of the Tn5 postcleavage complex and may mimic the DNA changes on paired complex formation. We have searched for a tryptophan residue in RAG1 that would be the functional equivalent of W298 in Tn5, which stabilizes the DNA interaction by stacking the flipped base on the indole ring. A W956A mutation in RAG1 had an inhibitory effect on both nicking and hairpin stages that could be rescued by abasic substrates. W956 is therefore a likely candidate for interacting with this base during hairpin formation.

Studies of V(D)J Recombination with Extrachromosomal Substrates

We have developed a series of new substrates to study the mechanism and regulation of lymphoid V(D)J recombination. The substrates remain extrachromosomal and are recovered and analyzed for recombination within one to two days after transfection into murine cells. Extrachromosomal substrates have several advantages over substrates that integrate into the genome. Because integrating substrates incorporate into the genome at different sites in each host cell, recombination may be variably influenced by different chromosomal contexts, thereby introducing an uncontrolled parameter into comparisons between different host lines or substrates. Also, a considerable length of time is required to establish cells with integrated exogenous DNA, and drug selection or cellular subcloning steps are necessary to isolate the recombinant clones. Quantitative studies are thus difficult or in many cases impossible.

Abnormal V(D)J Recombination in Murine Severe Combined Immune Deficiency: Absence of Coding Joints and Formation of Alternative Products

We have studied the VDJ recombination reaction in early B and T cells from mice homozygous for the severe combined immune deficiency (scid) mutation. Schuler et al. (1986) previously found abnormal deletions by Southern blot analysis at rearranged IgH and TCR β alleles in transformed B and T lymphocytes from seid mice. We have recently completed a study (Lieber et al. 1988a) in which we transfected scid lymphoid cell lines with VDJ recombination substrates that remain extrachromosomal. These substrates allow large numbers of recombinant reaction products to be collected (Hesse et al. 1987; Lieber et al. 1987), thereby permitting detailed analysis of the VDJ recombination reaction in scid lymphoid cells.

Chromatin Structure Near an Expressed Gene

The DNA within eukaryotic nuclei is complexed with basic proteins called histones to form a compact structure. Although little is known about the way in which higher orders of compaction are achieved, the two lowest levels of DNA packing in chromatin are relatively well understood. The fundamental chromatin subunit is the nucleosome. The central portion of each nucleosome is the chromatosome, which contains 165 base pairs (bp) of DNA wrapped in two superhelical turns about an octamer of histones. Each chromatosome is connected to its neighbor by a segment of linker DNA, the length of which varies from about 10 to 80 bp. The next level of compaction requires that the lineas polynucleosome filament be folded to form a fiber 30 nm in diameter. Most evidence suggests that this is a solenoidal structure in which the filament is supercoiled to give a fiber with about six nucleosomes per turn (Finch and Klug, 1976; Felsenfeld and McGhee, 1986).