DNA sequences at immunoglobulin switch region recombination sites.

The immunoglobulin heavy chain switch from synthesis of IgM to IgG, IgA or IgE is mediated by a DNA recombination event. Recombination occurs within switch regions, 2-10 kb segments of DNA that lie upstream of heavy chain constant region genes. A compilation of DNA sequences at more than 150 recombination sites within heavy chain switch regions is presented. Switch recombination does not appear to occur by homologous recombination. An extensive search for a recognition motif failed to find such a sequence, implying that switch recombination is not a site-specific event. A model for switch recombination that involves illegitimate priming of one switch region on another, followed by error-prone DNA synthesis, is proposed.     

The role of G-density in switch region repeats for immunoglobulin class switch recombination

The boundaries of R-loops are well-documented at immunoglobulin heavy chain loci in mammalian B cells. Within primary B cells or B cell lines, the upstream boundaries of R-loops typically begin early in the repetitive portion of the switch regions. Most R-loops terminate within the switch repetitive zone, but the remainder can extend a few hundred base pairs further, where G-density on the non-template DNA strand gradually drops to the genome average. Whether the G-density determines how far the R-loops extend is an important question. We previously studied the role of G-clusters in initiating R-loop formation, but we did not examine the role of G-density in permitting the elongation of the R-loop, after it had initiated. Here, we vary the G-density of different portions of the switch region in a murine B cell line. We find that both class switch recombination (CSR) and R-loop formation decrease significantly when the overall G-density is reduced from 46% to 29%. Short 50 bp insertions with low G-density within switch regions do not appear to affect either CSR or R-loop elongation, whereas a longer (150 bp) insertion impairs both. These results demonstrate that G-density is an important determinant of the length over which mammalian genomic R-loops extend.     

High fidelity extrachromosomal recombination and gene targeting in plants

The precision of extrachromosomal homologous recombination and gene targeting in plant cells was investigated. Recombination was directed to introns of selectable marker genes where potential changes could persist without affecting the function and therefore the selectability of the genes. Approximately 9 kb of crossover regions was rescued and sequenced. Changes were detected at a frequency below one point mutation per 1000 bp, indicating that extrachromosomal recombination and gene targeting both appear to occur with high fidelity.     

Copy choice mechanism of immunoglobulin heavy-chain switch recombination.

The immunoglobulin heavy-chain switch is mediated by a recombination event between DNA switch regions associated with donor and recipient constant-region genes. We have determined that the mutations which can be found in some switch regions after recombination appear to arise on only one strand of DNA. This result suggests that switch recombination involves error-prone synthesis of one DNA strand and ligation of the other strand from preexisting DNA.     

Homologous chromosome recombination generating immunoglobulin allotype and isotype switch variants.

We investigated whether spontaneous isotype switching in monoclonal antibody-producing hybridomas always occurs with genes on the same chromosome. Spleen cells of (BAB/ 25 X AKR/J) F1 mice, immunized with dansyl-keyhole limpet hemocyanin (DNS-KLH), were hybridized with NS-1 to generate hybridomas producing monoclonal anti-DNS antibodies of either the b or d haplotype of the BAB/25 or AKR/J parent, respectively. We selected isotype switch variants of such hybridomas using the fluorescence-activated cell sorter (FACS). Although in most cases the allotypic haplotype expressed by the parent and switch-variant hybridomas are the same, in one family of variants we noted a switch in haplotype along with the switch in isotype. This was noted in the selection of IgG2a switch variants from an IgG1 switch variant originally derived from an IgG3-producing parent. Biochemical and molecular studies confirm that the allotype switch variant expresses the same heavy-chain variable region gene complex as its parent hybridomas. As such, the allotype switch represents an example of spontaneous mitotic recombination between immunoglobulin heavy-chain genes, generating a single actively transcribed gene from loci previously positioned on different chromosomes.     

Non-B right-handed DNA conformations of homopurine.homopyrimidine sequences in the murine immunoglobulin C alpha switch region

The switch region of IgA immunoglobulin in mice cloned into a recombinant plasmid contains a supercoil-dependent S1 nuclease hypersensitive site, indicative of a non-B-DNA secondary structure. This site maps to the (AGGAG)28 direct repeat (DR2) of the alpha switch region and appears at a negative superhelical density of greater than 0.02. Studies with P1 nuclease and bromoacetaldehyde indicate that this structure is also present at neutral pH. S1 nuclease sensitivity is retained for the shorter repeat (AGGAG)6GA in a recombinant plasmid but is not seen for the repeat (CTGAG)6, corresponding to the DR1 repeat of the alpha switch region, or in a sequence corresponding to a portion of the consensus sequence which contains a short stretch of alternating pyrine-pyrimidine residues. Fine mapping of the (AGGAG)6GA and flanking sequences with dimethyl sulfate, bromoacetaldehyde, osmium tetroxide, and diethyl pyrocarbonate reveals an asymmetric pattern of modification dependent on both pH and supercoiling. Two-dimensional gel electrophoresis at low pH shows the relaxation of 3 superhelical turns on formation of this structure by the (AGGAG)6GA repeat. These results are most consistent with the formation of an intramolecular triple-strand.     

Regulation of DNA end joining, resection, and immunoglobulin class switch recombination by 53BP1

53BP1 is a DNA damage protein that forms phosphorylated H2AX (γ-H2AX) dependent foci in a 1 Mb region surrounding DNA double-strand breaks (DSBs). In addition, 53BP1 promotes genomic stability by regulating the metabolism of DNA ends. We have compared the joining rates of paired DSBs separated by 1.2 kb to 27 Mb on chromosome 12 in the presence or absence of 53BP1. 53BP1 facilitates joining of intrachromosomal DSBs but only at distances corresponding to γ-H2AX spreading. In contrast, DNA end protection by 53BP1 is distance independent. Furthermore, analysis of 53BP1 mutants shows that chromatin association, oligomerization, and N-terminal ATM phosphorylation are all required for DNA end protection and joining as measured by immunoglobulin class switch recombination. These data elucidate the molecular events that are required for 53BP1 to maintain genomic stability and point to a model wherein 53BP1 and H2AX cooperate to repress resection of DSBs.     

Nucleic acid structures and enzymes in the immunoglobulin class switch recombination mechanism

Class switch recombination is the gene rearrangement process by which our B lymphocytes change from IgM production to IgG, IgA, or IgE. Unlike the well-characterized V(D)J recombination, the mechanism of class switch recombination has been largely enigmatic until very recent progress has begun to shed light on this gene rearrangement process. Progress has been made on the enzymes involved in leading to the DNA cleavage events and on identifying the unusual DNA structures that those enzymes recognize.     

Repetitive sequences in class-switch recombination regions of immunoglobulin heavy chain genes

Immunoglobulin class switch involves a unique recombination event that takes place at the region 5′ to each heavy chain constant region gene during B lymphocyte differentiation. Such regions that are responsible for the class-switch recombination are defined as S regions (Kataoka et al., Proc. Natl. Acad. Sci. USA 77, 919, 1980). We have cloned a rearranged γ2b gene from a mouse myeloma (MPC11) and compared its structure with the germ line counterparts. The rearranged γ2b gene contained the 5′ flanking region of the γ3 gene (S_γ3 region) which are linked to the 5′ flanking region of the γ2b gene (S_γ2b region). We have determined nucleotide sequences surrounding the recombination site of the rearranged and germ line γ2b genes, which include the S_γ2b and S_γ3 regions. Both _γ2b and S_γ3 regions comprise tandem repetition of conserved units of 49 bp. Similar 49 bp repeating units are also found in the previously determined sequence of the S_γ1 region in which class-switch recombination took place in MC101 myeloma. The nucleotide sequences of the S_γ1, S_γ2b and S_γ3 repeating units share significant homology with each other. The Sμ region, partial nucleotide sequence of which was previously determined, contains abundant short sequences such as AGCT, TGGG and AGCTGGGG which are shared in common by repeating sequences in S_γ regions. These results suggest that the recombination responsible for class switch from μ to γ or from a γ to another γ, may be facilitated directly or indirectly by homology of repeating sequences in S regions.     

Rapid assay for extrachromosomal homologous recombination in monkey cells.

Most of the recombination assays based on the regeneration of selectable marker genes after transient infection or stable integration of DNA into mammalian cells are time consuming. We have used plasmids containing two truncated but overlapping segments of the neomycin resistance gene to rapidly quantitate and characterize the time course of extrachromosomal homologous recombination of DNA transfected into monkey COS cells. By transiently infecting cells with these recombination substrates, extracting Hirt DNA after 1 to 4 days, and transforming recombination-deficient Escherichia coli, we have shown that recombination between direct repeats occurs at frequencies of 1 to 4%. We have also used Southern blot analysis to directly characterize the recombination of this DNA in COS cells and to demonstrate that double-strand breaks in the region of homology increase recombination frequencies 10- to 50-fold.     

Regulation of Staphylococcus protease using complement, interferon and immunoglobulin as substrates.

The effects of various agents on the cleavage of serum albumin, interferon, immunoglobulin and complement component C1q by the extracellular protease from Staphylococcus aureus were analysed by SDS-polyacrylamide gel electrophoresis. Arachidonic acid moderately stimulated the proteolysis of serum albumin, interferon and complement component. Phosphatidic acid effectively enhanced the proteolysis of serum albumin and IgG, whereas it inhibited the cleavage of IgM. The proteolysis of IgG was appreciably enhanced by sphingosine. In contrast, phosphatidyl choline and phosphatidyl glycerol were shown to have an inhibitory effect on the proteolysis of IgG and IgM. Phosphatidyl serine, phosphatidyl inositol and phosphatidyl ethanolamine also inhibited the proteolysis of IgG. The failure of any of these agents to exert a persistent effect on the cleavage of all substrates, revealed the complexity of the interactions among the agent, the substrate and the protease.     

The block in immunoglobulin class switch recombination caused by activation-induced cytidine deaminase deficiency occurs prior to the generation of DNA double strand breaks in switch mu region

Affinity maturation of the Ab repertoire in germinal centers leads to the selection of high affinity Abs with selected heavy chain constant regions. Ab maturation involves two modifications of the Ig genes, i.e., somatic hypermutation and class switch recombination. The mechanisms of these two processes are not fully understood. As shown by the somatic hypermutation and class switch recombination-deficient phenotype of activation-induced cytidine deaminase (AID)-deficient patients (hyperIgM type 2 syndrome) and mice, both processes require the AID molecule. Somatic DNA modifications require DNA breaks, which, at least for class switch recombination, lead to dsDNA breaks. By using a ligation-mediated PCR, it was found that class switch recombination-induced dsDNA breaks in S mu switch regions were less frequent in AID-deficient B cells than in AID-proficient B cells, thus indicating that AID acts upstream of DNA break induction.     

Differential effects of Rad52p overexpression on gene targeting and extrachromosomal homologous recombination in a human cell line

Overexpression of the RAD52 epistasis group of gene products is a convenient way to investigate their in vivo roles in homologous recombination (HR) and DNA repair. Overexpression has the further attraction that any associated stimulation of HR may facilitate gene-targeting applications. Rad51p or Rad52p overexpression in mammalian cells have previously been shown to enhance some forms of HR and resistance to ionising radiation, but the effects of Rad52p overexpression on gene targeting have not been tested. Here we show that Rad52p overexpression inhibits gene targeting while stimulating extrachromosomal HR. We also find that Rad52p overexpression affects cell-cycle distribution, impairs cell survival and is lost during extensive passaging. Therefore, we suggest that excess Rad52p can inhibit the essential RAD51-dependent pathways of HR most likely to be responsible for gene targeting, while at the same time stimulating the RAD51-independent pathway thought to be responsible for extrachromosomal HR. The data also argue against Rad52p overexpression as a means of promoting gene targeting, and highlight the limitations of using a single HR assay to assess the overall status of HR.