The mechanism of osmotic transfection of avian embryonic erythrocytes: analysis of a system for studying developmental gene expression.

We have undertaken a study of the mechanism of DNA transfer into primary chicken erythrocytes by a method named osmotic transfection. The cells are subjected to controlled osmotic swelling in NH4Cl and then ruptured in a lower osmotic strength solution containing DNA and DEAE-dextran. The osmotic rupture results in transient formation of a single hole in the cell membrane, which is followed within hours by recovery of near normal levels of RNA and protein synthesis. The association of DNA with the cells is much greater for ruptured than for unruptured cells or for cells that have been lysed and resealed before DNA is added. Transient formation of pores in the cell membrane is apparently essential for high rates of macromolecular transfer into the cell. DEAE-dextran increases the amount of DNA associated with the cells, especially after cell rupture. Our understanding of the mechanism has allowed us to extend the application of osmotic transfection to essentially all developmental stages of avian erythroid differentiation. Osmotic transfections were done with plasmids containing the chloramphenicol acetyl transferase (cat) gene placed between the chicken beta-globin promoter and the 3' beta-globin enhancer. The pattern of CAT expression at sequential developmental stages parallels that of the endogenous gene, showing that osmotically transfected cells appear to retain developmental fidelity. The approach provides a convenient, sensitive, and flexible system for the study of transient gene expression as a function of development.

Stable RNA/DNA hybrids in the mammalian genome: inducible intermediates in immunoglobulin class switch recombination.

Although it is well established that mammalian class switch recombination is responsible for altering the class of immunoglobulins, the mechanistic details of the process have remained unclear. Here, we show that stable RNA/DNA hybrids form at class switch sequences in the mouse genome upon cytokine-specific stimulation of class switch in primary splenic B cells. The RNA hybridized to the switch DNA is transcribed in the physiological orientation. Mice that constitutively express an Escherichia coli ribonuclease H transgene show a marked reduction in RNA/DNA hybrid formation, an impaired ability to generate serum immunoglobulin G antibodies, and significant inhibition of class switch recombination in their splenic B cells. These data provide evidence that stable RNA/DNA hybrids exist in the mammalian nuclear genome, can serve as intermediates for physiologic processes, and are mechanistically important for efficient class switching in vivo.

Restoration of X-ray resistance and V(D)J recombination in mutant cells by Ku cDNA.

Three genetic complementation groups of rodent cells are defective for both repair of x-ray-induced double-strand breaks and V(D)J recombination. Cells from one group lack a DNA end-binding activity that is biochemically and antigenically similar to the Ku autoantigen. Transfection of complementary DNA (cDNA) that encoded the 86-kilodalton subunit of Ku rescued these mutant cells for DNA end-binding activity, x-ray resistance, and V(D)J recombination activity. These results establish a role for Ku in DNA repair and recombination. Furthermore, as a component of a DNA-dependent protein kinase, Ku may initiate a signaling pathway induced by DNA damage.

Superinduction of endogenous type C virus by 5-bromodeoxyuridine from transformed mouse clones.

Certain transformed subclones of the mouse cell line BALB/3T3 release 5 to 15 times more type C virus per cell than the parent cell line after treatment with 5-bromodeoxyuridine. Virus release begins within 8 hours and exponentially increases for the first 24 to 48 hours. Superinducibility is associated with the transformed phenotype.

Mammalian cells in culture frequently release type C viruses.

Cell cultures commonly used in animal cell research, both cell strains and continuous cell lines from various mammalian species, spontaneously produce type C RNA viruses.

Bovine leukemia virus genes in the DNA of leukemic cattle.

Reverse transcripts of the rna genome of the bovine leukemia virus (BLV) as well as 125I-labeled BLV RNA hybridize to the DNA of tissues from leukemic cattle with the adult form of the disease but not to bovine thymic lymphoma or normal bovine tissues.

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.

Tying loose ends: roles of Ku and DNA-dependent protein kinase in the repair of double-strand breaks.

A convergence of information from biochemistry, yeast and mammalian genetics, immunology, and radiation biology has permitted identification of some of the protein participants - Ku, DNA-PK, XRCC4 - and the reaction intermediates in DNA end joining, suggesting how broken chromosomal ends may be recognized and repaired in eukaryotic cells. Some components may be defective in inherited disorders.

Immunoglobulin diversity: rearranging by cutting and repairing.

The recombination process that assembles antigen-receptor genes is now understood in some biochemical detail. The initial steps reflect a common theme seen in retroviral integration and prokaryotic transposition, and the later steps involve the enzymatic machinery for double-strand break DNA repair.

Antibody diversity: a link between switching and hypermutation.

Recent work indicates that mutations in a cytidine deaminase homologue ablate both immunoglobulin class switch recombination and somatic hypermutation. These findings now explain cases of autosomal hyper-IgM syndrome and reveal that critical components for key functions of B cells require RNA editing.

DNA ligase IV binds to XRCC4 via a motif located between rather than within its BRCT domains.

The covalent rejoining of DNA ends at single-stranded or double-stranded DNA breaks is catalyzed by DNA ligases. Four DNA ligase activities (I-IV) have been identified in mammalian cells [1]. It has recently been demonstrated that DNA ligase IV interacts with and is catalytically stimulated by the XRCC4 protein [2,3], which is essential for DNA double-strand break repair and the genomic rearrangement process of V(D)J recombination [4]. Together with the finding that the yeast DNA ligase IV homologue is essential for nonhomologous DNA end joining [5-7], this has led to the hypothesis that mammalian DNA ligase IV catalyzes ligation steps in both of these processes [8]. DNA ligase IV is characterized by a unique carboxy-terminal tail comprising two BRCT (BRCA1 carboxyl terminus) domains. BRCT domains were initially identified in the breast cancer susceptibility protein BRCA1 [9], but are also found in other DNA repair proteins [10]. It has been suggested that DNA ligase IV associates with XRCC4 via its tandem BRCT domains and that this may be a general model for protein-protein interactions between DNA repair proteins [3]. We have performed a detailed deletional analysis of DNA ligase IV to define its XRCC4-binding domain and to characterize regions essential for its catalytic activity. We find that a region in the carboxy-terminal tail of DNA ligase IV located between rather than within BRCT domains is necessary and sufficient to confer binding to XRCC4. The catalytic activity of DNA ligase IV is affected by mutations within the first two-thirds of the protein including a 67 amino-acid amino-terminal region that was previously thought not to be present in human DNA ligase IV [11].

The nonhomologous DNA end joining pathway is important for chromosome stability in primary fibroblasts.

There are two types of chromosome instability, structural and numerical, and these are important in cancer. Many structural abnormalities are likely to involve double-strand DNA (dsDNA) breaks. Nonhomologous DNA end joining (NHEJ) and homologous recombination are the major pathways for repairing dsDNA breaks. NHEJ is the primary pathway for repairing dsDNA breaks throughout the G0, G1 and early S phases of the cell cycle [1]. Ku86 and DNA ligase IV are two major proteins in the NHEJ pathway. We examined primary dermal fibroblasts from mice (wild type, Ku86(+/-), Ku86(-/-), and DNA ligase IV(+/-)) for chromosome breaks. Fibroblasts from Ku86(+/-) or DNA ligase IV(+/-) mice have elevated frequencies of chromosome breaks compared with those from wild-type mice. Fibroblasts from Ku86(-/-) mice have even higher levels of chromosome breaks. Primary pre-B cells from the same animals did not show significant accumulation of chromosome breaks. Rather the pre-B cells showed increased cell death. These studies demonstrate that chromosome breaks arise frequently and that NHEJ is required to repair this constant spontaneous damage.

Antigen receptor gene rearrangement.

Two specialized forms of site-directed double-strand (ds) DNA breakage and rejoining are part of the physiologic program of lymphocytes. One is recombination of the V, D and J gene sequences, termed V(D)J recombination, occurring during early B- and T-cell development, and the other is class-switch recombination occurring exclusively in mature B cells. For V(D)J recombination significant progress has been made recently elucidating the biochemistry of the reaction. In particular our understanding of how DNA ds breaks are both generated and rejoined has increased. For class-switch recombination no definitive information is known about the nucleases required for making the ds breaks, but recent evidence suggests that the joining phase shares activities also required for V(D)J recombination and general DNA ds break repair.

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.

V(D)J recombination: signal and coding joint resolution are uncoupled and depend on parallel synapsis of the sites.

V(D)J recombination in lymphoid cells is a site-specific process in which the activity of the recombinase enzyme is targeted to signal sequences flanking the coding elements of antigen receptor genes. The order of the steps in this reaction and their mechanistic interdependence are important to the understanding of how the reaction fails and thereby contributes to genomic instability in lymphoid cells. The products of the normal reaction are recombinant joints linking the coding sequences of the receptor genes and, reciprocally, the signal ends. Extrachromosomal substrate molecules were modified to inhibit the physical synapsis of the recombination signals. In this way, it has been possible to assess how inhibiting the formation of one joint affects the resolution efficiency of the other. Our results indicate that signal joint and coding joint formation are resolved independently in that they can be uncoupled from each other. We also find that signal synapsis is critical for the generation of recombinant products, which greatly restricts the degree of potential single-site cutting that might otherwise occur in the genome. Finally, inversion substrates manifest synaptic inhibition at much longer distances than do deletion substrates, suggesting that a parallel rather than an antiparallel alignment of the signals is required during synapsis. These observations are important for understanding the interaction of V(D)J signals with the recombinase. Moreover, the role of signal synapsis in regulating recombinase activity has significant implications for genome stability regarding the frequency of recombinase-mediated chromosomal translocations.

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.

Protein-protein and protein-DNA interaction regions within the DNA end-binding protein Ku70-Ku86.

DNA ends are generated during double-strand-break repair and recombination. A p70-p86 heterodimer, Ku, accounts for the DNA end binding activity in eukaryotic cell extracts. When one or both subunits of Ku are missing, mammalian cells are deficient in double-strand-break repair and in specialized recombination, such as V(D)J recombination. Little is known of which regions of Ku70 and Ku86 bind to each other to form the heterodimeric complex or of which regions are important for DNA end binding. We have done genetic and biochemical studies to examine the domains within the two subunits important for protein assembly and for DNA end binding. We found that the C-terminal 20-kDa region of Ku70 and the C-terminal 32-kDa region of Ku86 are important for subunit-subunit interaction. For DNA binding, full-length individual subunits are inactive, indicating that heterodimer assembly precedes DNA binding. DNA end binding activity by the heterodimer requires the C-terminal 40-kDa region of Ku70 and the C-terminal 45-kDa region of Ku86. Leucine zipper-like motifs in both subunits that have been suggested as the Ku70-Ku86 interaction domains do not appear to be the sites of such interaction because these are dispensable for both assembly and DNA end binding. On the basis of these studies, we have organized Ku70 into nine sequence regions conserved between Saccharomyces cerevisiae, Drosophila melanogaster, mice, and humans; only the C-terminal three regions are essential for assembly (amino acids [aa] 439 to 609), and the C-terminal four regions appear to be essential for DNA end binding (aa 254 to 609). Within the minimal active fragment of Ku86 necessary for subunit interaction (aa 449 to 732) and DNA binding (aa 334 to 732), a proline-rich region is the only defined motif.

Mechanistic basis for coding end sequence effects in the initiation of V(D)J recombination.

V(D)J recombination is directed by recombination signal sequences. However, the flanking coding end sequence can markedly affect the frequency of the initiation of V(D)J recombination in vivo. Here we demonstrate that the coding end sequence effect can be qualitatively and quantitatively recapitulated in vitro with purified RAG proteins. We find that coding end sequence specifically affects the nicking step, which is the first biochemical step in RAG-mediated cleavage. The subsequent hairpin formation step is not affected by the coding end sequence. Furthermore, the coding end sequence effect can be ablated by prenicking the substrate, indicating that the coding end effect is specific to the nicking step. In reactions in which both 12- and 23-substrates are present, a suboptimal coding end sequence on one signal can slow down hairpin formation at the partner signal, a result consistent with models in which coordination between the signals occurs at the hairpin formation step. The coding end sequence effect on nicking and the coupling of the 12- and 23-substrates explains how hairpin formation can be rate limiting for some 12/23 pairs, whereas nicking can be rate limiting when low-efficiency coding end sequences are involved.

The RAG-HMG1 complex enforces the 12/23 rule of V(D)J recombination specifically at the double-hairpin formation step.

A central unanswered question concerning the initial phases of V(D)J recombination has been at which step the 12/23 rule applies. This rule, which governs which variable (V), diversity (D), and joining (J) segments are able to pair during recombination, could operate at the level of signal sequence synapsis after RAG-HMG1 complex binding, signal nicking, or signal hairpin formation. It has also been unclear whether additional proteins are required to achieve adherence to the 12/23 rule. We developed a novel system for the detailed biochemical analysis of the 12/23 rule by using an oligonucleotide-based substrate that can include two signals. Under physiologic conditions, we found that the complex of RAG1, RAG2, and HMG1 can successfully recapitulate the 12/23 rule with the same specificity as that seen intracellularly and in crude extracts. The cleavage complex can bind and nick 12x12 and 23x23 substrates as well as 12x23 substrates. However, hairpin formation occurs at both of the signals only on 12x23 substrates. Moreover, under physiologic conditions, the presence of a partner 23-bp spacer suppresses single-site hairpin formation at a 12-bp spacer and vice versa. Hence, this study illustrates that synapsis suppresses single-site reactions, thereby explaining the high physiologic ratio of paired versus unpaired V(D)J recombination events in lymphoid cells.

The nicking step in V(D)J recombination is independent of synapsis: implications for the immune repertoire.

In all of the transposition reactions that have been characterized thus far, synapsis of two transposon ends is required before any catalytic steps (strand nicking or strand transfer) occur. In V(D)J recombination, there have been inconclusive data concerning the role of synapsis in nicking. Synapsis between two 12-substrates or between two 23-substrates has not been ruled out in any studies thus far. Here we provide the first direct tests of this issue. We find that immobilization of signals does not affect their nicking, even though hairpinning is affected in a manner reflecting its known synaptic requirement. We also find that nicking is kinetically a unireactant enzyme-catalyzed reaction. Time courses are no different between nicking seen for a 12-substrate alone and a reaction involving both a 12- and a 23-substrate. Hence, synapsis is neither a requirement nor an effector of the rate of nicking. These results establish V(D)J recombination as the first example of a DNA transposition-type reaction in which catalytic steps begin prior to synapsis, and the results have direct implications for the order of the steps in V(D)J recombination, for the contribution of V(D)J recombination nicks to genomic instability, and for the diversification of the immune repertoire.