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.     

Mechanism of meiotic recombination in eukaryotes—A theory

It is proposed that in meiotic chromosomes single strand breaks of DNA originate either in the delayed regions of replicons or as a result of the excision activity of DNA polymerase during zygotene DNA synthesis. Rejoining of the break points belonging to non-sister chromatids takes place by switching over of the polymerase from one strand of DNA to another non-sister strand of the same polarity and gives rise to recombination intermediates (half-chromatid chiasmata). Strand migration in a recombination intermediate or copying of the same parental strand twice during zygotene as a consequence of a delay in copying the homologous strand would lead to gene conversion. Nicking of the cross strands (parental strands) in any recombination intermediate and subsequent repair leads to recombination for flanking markers. A possible way in which three-strand double crossovers occur and the process of recombination are discussed.     

Mismatch repair converts AID-instigated nicks to double-strand breaks for antibody class-switch recombination

Mismatch repair (MMR) proteins are important for antibody class-switch recombination (CSR), but their roles are unknown. We propose a model for the function of MMR in CSR in which MMR proteins convert single-strand nicks instigated by activation-induced cytidine deaminase (AID) into the double-strand breaks (DSBs) that are required for CSR. This model does not invoke any novel functions for MMR but simply posits that, owing to numerous single-strand nicks in the switch (S) regions of both DNA strands, when MMR proteins are recruited by U:G mismatches, they excise one strand of DNA and soon reach a nick on the opposite strand. This halts excision activity and creates a DSB. This model explains why B cells that lack either S mu and MSH2 or UNG and MSH2 cannot undergo CSR.     

Schizosaccharomyces pombe switches mating type by the synthesis-dependent strand-annealing mechanism

Schizosaccharomyces pombe cells can switch between two mating types, plus (P) and minus (M). The change in cell type occurs due to a replication-coupled recombination event that transfers genetic information from one of the silent-donor loci, mat2P or mat3M, into the expressed mating-type determining mat1 locus. The mat1 locus can as a consequence contain DNA encoding either P or M information. A molecular mechanism, known as synthesis-dependent strand annealing, has been proposed for the underlying recombination event. A key feature of this model is that only one DNA strand of the donor locus provides the information that is copied into the mat1. Here we test the model by constructing strains that switch using two different mutant P cassettes introduced at the donor loci, mat2 and mat3. We show that in such strains wild-type P-cassette DNA is efficiently generated at mat1 through heteroduplex DNA formation and repair. The present data provide an in vivo genetic test of the proposed molecular recombination mechanism.     

Products and implied mechanism of H chain switch recombination

The Ig H chain switch is a DNA recombination event. The recombination occurs between two or more switch regions, areas of tandem sequence duplication that lie upstream of the corresponding H chain C region genes. We have determined the DNA sequence at four recombination sites in three molecularly cloned, rearranged switch regions. All eight donor and recipient recombination sites are at the common pentamers GGGGT, GAGCT, and GGTGG. One of the switch recombination events is an inversion of S gamma 3 sequences. Another of the recombinational events is an internal S gamma 1 deletion, which may be switch enzyme mediated. These results, together with other switch recombination site sequences, suggest that switch recombination is mediated by cutting enzymes with modest specificity and religation enzymes with no specificity.     

Mutations, duplication, and deletion of recombined switch regions suggest a role for DNA replication in the immunoglobulin heavy-chain switch.

The heavy-chain switch from immunoglobulin M (IgM) expression to IgA expression is mediated by a recombination event between segments of DNA called switch regions. The switch regions lie two to six kilobases upstream of the mu and alpha constant region coding segments. Switch recombination to IgA expression results in a recombinant mu-alpha switch region upstream of the expressed alpha constant region gene. We have characterized the products of switch recombination by a lymphoma cell line, I.29. Two sets of molecular clones represent the expected products of simple mu to alpha switches. Five members of a third set of molecular clones share the same recombination site in both the mu and the alpha switch regions, implying that the five molecular clones were derived from a single switch recombination event. Surprisingly, the five clones fall into two sets of sequences, which differ from each other by several point mutations and small deletions. Duplication of switch region sequences are also found in these five molecular clones. An explanation for these data is that switch recombination involves DNA synthesis, which results in nucleotide substitutions, small deletions, and duplications.     

Reduced switching in SCID B cells is associated with altered somatic mutation of recombined S regions

Deoxyribonucleic acid double-stranded breaks act as intermediates in Ig V(D)J recombination and probably perform a similar function in class switch recombination between IgH C genes. In SCID mice, V(D)J recombination is blocked because the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) protein is defective. We show in this study that switching to all isotypes examined was detectable when the SCID mutation was introduced into anti-hen egg lysozyme transgenic B cells capable of undergoing class switch recombination, but switching was significantly reduced in comparison with control B cells of the same specificity lacking the RAG1 gene. Thus, DNA-PKcs is involved in switching to all isotypes, but plays a lesser role in the switching process than it does in V(D)J-coding joint formation. The higher level of switching observed by us in SCID B cells compared with that observed by others in DNA-PKcs(null) cells raises the possibility that kinase-deficient DNA-PKcs can function in switching. Point mutation of G:C base pairs with cytidines on the sense strand was greatly reduced in recombined switch regions from SCID cells compared with control RAG1(-/-) B cells. The preferential loss of sense strand cytidine mutations from hybrid S regions in SCID cells suggests the possibility that nicks might form in S regions of activated B cells on the template strand independently of activation-induced cytidine deaminase and are converted to double-strand breaks when activation-induced cytidine deaminase deaminates the non-template strand.     

The immunoglobulin heavy chain switch: structural features of gamma 1 recombinant switch regions

The immunoglobulin heavy chain isotype switch is mediated by a DNA rearrangement involving specific genomic segments referred to as switch regions. Switch regions are composed of tandemly repeated simple sequences. The role of the tandemly repeated structure of switch regions in the switch recombination process is not understood. We mapped eight recombination sites--six in the gamma 1 and two in the gamma 3 tandem arrays. In addition, we obtained molecular clones representing three of the six gamma 1 rearrangements, and determined the nucleotide sequences of the recombination sites in each. In general, the rearrangements are confined to the tandem repeat units, and are not clustered in a particular portion of either the gamma 3 or gamma 1 switch region. Nucleotide sequence analysis of one of the recombinant clones, gamma M35, reveals evidence for a successive switch event wherein a recombination between S mu and S gamma 3 was followed by recombination 57 bp downstream with S gamma 1. gamma 1 sequence data from the molecular clones we obtained, together with similar data from other investigators regarding the gamma 1, gamma 2b, and gamma 2a switch regions, reveals that recombinations tend to occur at homologous positions of the respective gamma-unit repeats, adjacent to the elements AGCT and GGGG found in each. This finding suggests that the cutting and religation step of the recombination process is mediated by a recombinase common to the four gamma-isotypes.     

Analysis of immunoglobulin Sgamma3 recombination breakpoints by PCR: implications for the mechanism of isotype switching.

The molecular mechanism of immunoglobulin switch recombination is poorly understood. Switch recombination occurs between pairs of switch regions located upstream of the constant heavy chain genes. Previously we showed that switch recombination breakpoints cluster to a defined subregion in the Sgamma3, Sgamma1 and Sgamma2b tandem repeats. We have developed a strategy for direct amplification of Smu/Sgamma3 composite fragments as well as Smu and Sgamma3 regions by PCR. This assay has been used to analyze the organization of Smu, Sgamma3 and a series of Smu/Sgamma3 recombination breakpoints from hybridomas and normal mitogen-activated splenic B cells. DNA sequence analysis of the switch fragments showed direct joining of Smu and Sgamma3 without deletions or duplications. Mutations were found in two switch junctions on both sides of the crossover point, suggesting that template switching is the most likely model for the mechanism of switch recombination. Statistical analysis of the positions of the recombination breakpoints in the Sgamma3 tandem repeat indicates the presence of two sub-clusters, suggesting non-random usage of DNA substrate in the recombination reaction.     

53BP1-mediated DNA double strand break repair: insert bad pun here

53BP1 is an established player in the cellular response to DNA damage and is a canonical component of ionizing-radiation induced foci--that cadre of proteins which assemble at DNA double strand breaks following radiation exposure and which are readily visualized by immunofluorescence microscopy. While its roles in p53 regulation and cell cycle checkpoint activation have been studied for some time, the impact of 53BP1 on DNA double strand break rejoining has only come to light in the past few years. Convincing evidence now exists for 53BP1 significantly affecting the outcome of DNA double strand break repair in several contexts, many of which hint to an important role in modulating chromatin structure surrounding the break site. Here, we highlight the known and emerging roles of 53BP1 in DNA double strand break repair, including the repair of lesions induced within heterochromatin, following telomere uncapping, in long-range V(D)J recombination, during immunoglobulin class switch recombination and its much debated role in regulating resection during homologous recombination.     

Construction and evaluation of a kinetic scheme for RecA-mediated DNA strand exchange

The Escherichia coli RecA protein is the prototype of a class of proteins playing a central role in genomic repair and recombination in all organisms. The unresolved mechanistic strategy by which RecA aligns a single strand of DNA with a duplex DNA and mediates a DNA strand switch is central to understanding its recombinational activities. Toward a molecular-level understanding of RecA-mediated DNA strand exchange, we explored its mechanism using oligonucleotide substrates and the intrinsic fluorescence of 6-methylisoxanthopterin (6MI). Steady- and presteady-state spectrofluorometric data demonstrate that the reaction proceeds via a sequential four-step mechanism comprising a rapid, bimolecular association step followed by three slower unimolecular steps. Previous authors have proposed multistep mechanisms involving two or three steps. Careful analysis of the differences among the experimental systems revealed a previously undiscovered intermediate (N1) whose formation may be crucial in the kinetic discrimination of homologous and heterologous sequences. This observation has important implications for probing the fastest events in DNA strand exchange using 6MI to further elucidate the molecular mechanisms of recombination and recombinational repair.     

The RecA protein as a recombinational repair system

The Escherichia coli RecA protein plays a central role in homologous genetic recombination, recombinational repair, and several other processes in bacteria. In vitro, an extended filament involving thousands of RecA monomers promotes a reaction in which individual DNA strands switch pairing partners (DNA strand exchange). This reaction has been extensively studied as a paradigm for the central steps in recombination. Because the strand-exchange reaction is relatively simple and isoenergetic, the complexity of the RecA system that carries it out has led to controversy about the functional significance of many fundamental properties of RecA. Filamentous protein structures involving thousands of RecA monomers, which hydrolyse 100 ATPs per base pair of heteroduplex DNA formed, are hard to rationalize in the context of recombination between two homologous DNAs. The thermodynamic barriers to strand exchange are much too small. These molecular features of the system can be easily rationalized, however, by shifting the focus to DNA repair.