Mechanistic properties of immunoglobulin somatic mutation.
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Animals↗
Biomedical subjects
M R Lieber
Publications and source records attributed to M R Lieber.
Lagging strand DNA synthesis at the eukaryotic replication fork involves binding and stimulation of FEN-1 by proliferating cell nuclear antigen.
The 5'-->3'-exonuclease domain of Escherichia coli DNA polymerase I is required for the completion of lagging strand DNA synthesis, and yet this domain is not present in any of the eukaryotic DNA polymerases. Recently, the gene encoding the functional and evolutionary equivalent of this 5'-->3'-exonuclease domain has been identified. It is called FEN-1 in mouse and human cells and RTH1 in Saccharomyces cerevisiae. This 42-kDa enzyme is required for Okazaki fragment processing. Here we report that FEN-1 physically interacts with proliferating cell nuclear antigen (PCNA), the processivity factor for DNA polymerases delta and epsilon. Through protein-protein interactions, PCNA focuses FEN-1 on branched DNA substrates (flap structures) and on nicked DNA substrates, thereby stimulating its activity 10-50-fold but only if PCNA can functionally assemble as a toroidal trimer around the DNA. This interaction is important in the physical orchestration of lagging strand synthesis and may have implications for how PCNA stimulates other members of the FEN-1 nuclease family in a broad range of DNA metabolic transactions.
Base Sequence↗
Strand specificity in the transcriptional targeting of recombination at immunoglobulin switch sequences.
B-lymphocyte-specific class switch recombination is known to occur between pairs of 2- to 10-kb switch regions located immediately upstream of the immunoglobulin constant heavy-chain genes. Others have shown that the recombination is temporally correlated with the induction of transcription at the targeted switch regions. To determine whether this temporal correlation is due to a mechanistic linkage, we have developed an extrachromosomal recombination assay that closely recapitulates DNA deletional class switch recombination. In this assay, the rate of recombination is measured between 24 and 48 hr posttransfection. We find that recombinants are generated in a switch sequence-dependent manner. Recombination occurs with a predominance within B-cell lines representative of the mature B-cell stage and within a subset of pre-B-cell lines. Transcription stimulates the switch sequence-dependent recombination. Importantly, transcription activates recombination only when directed in the physiologic orientation but has no effect when directed in the nonphysiologic orientation.
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The scid factor on human chromosome 8 restores V(D)J recombination in addition to double-strand break repair.
The murine severe combined immune deficiency mutation (scid) is characterized by a lack of B- and T-lymphoid cells due to a defect in lymphoid V(D)J recombination. Moreover, defective rejoining of DNA double-strand breaks (dsb) in scid cells also results in a marked increase in sensitivity to ionizing radiation. Recently, the putative human homologue of the murine scid gene locus, HYRC1, was assigned to human chromosome 8q11, based on the radiation sensitivity of scid cells as compared to scid:human cell hybrids carrying portions of human chromosome 8. Given the precedent (e.g., ataxia-telangiectasia) for genes other than the affected one being able to complement radiation defects, we were interested in determining if the V(D)J recombination defect was also corrected by the HYRC1 locus. The V(D)J recombination analysis using extrachromosomal DNA substrates in control scid cells (SC3VA2) versus complemented cells (RD13B2) indicates that the radiation sensitivity-complemented cells (RD13B2) are also fully complemented for the V(D)J recombination reaction, whereas the control (uncomplemented) cells (SC3VA2) fail to carry out V(D)J recombination normally. Slightly over 60% of the radiation-induced dsb are rejoined even in scid cells, and this alternative pathway is temperature sensitive. Only the remaining 30-35% of dsb require the introduction of the HYRC1 locus, and this pathway is not temperature sensitive. This merely partial contribution of the scid factor to the repair process suggests the presence of another pathway of dsb repair. Our results indicate that the HYRC1 locus, assigned to human chromosome 8q11, encodes the scid factor, which is involved in all V(D)J recombination coding joint formation and in 30-35% of dsb repair by the temperature-resistant pathway.
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DNA structural elements required for FEN-1 binding.
In eukaryotic cells, a 5'-flap DNA endonuclease and a double-stranded DNA 5'-exonuclease activity reside within a 42-kDa enzyme called FEN-1 (flap endonuclease-1 and 5(five)'-exonuclease). This endo/exonuclease has been shown to be highly homologous to human XP-G, Saccharomyces cerevisiae RAD2, and S. cerevisiae YKL510. Like FEN-1, these related structure-specific nucleases recognize and cleave a branched DNA structure called a DNA flap and its derivative, called a pseudo Y-structure. To dissect the important structural components of the DNA flap structure, we have developed a mobility shift assay. We find that the Fadj strand (located adjacent to the displaced flap strand) is necessary for efficient binding and cleavage of flap structures by FEN-1. When this strand is absent or when it is present, but recessed from the elbow of the flap strand, binding efficiency drops. Further investigation of the role of the Fadj strand using double flap structures reveals that the Fadj strand is necessary to provide a double-stranded template upon which FEN-1 can bind near the elbow of the flap strand. These results provide a basis for understanding how this structure-specific nuclease recognizes a variety of DNA substrates.
Base Sequence↗
Sequence of human FEN-1, a structure-specific endonuclease, and chromosomal localization of the gene (FEN1) in mouse and human.
We recently purified and cloned the gene for a DNA structure-specific endonuclease, FEN-1, from murine cells. The murine protein recognizes 5' DNA flap structures that have been proposed in DNA replication, repair, and recombination. Here, we report the sequence of the human FEN1 gene. The translated sequence is identical to peptide sequence obtained from maturation factor-1, which is 1 of the 10 essential proteins for cell-free DNA replication. The human protein has the same structure-specific DNA endonuclease activity as the murine protein. Two human chromosomal hybridization signals, 11q12 and 1p22.2, were observed by FISH analysis using human genomic clones homologous to the mouse Fen-1 gene. The localization on human 11q12 was confirmed using radiation-reduced hybrids. The mouse Fen-1 gene is assigned to chromosome 19 based on somatic cell hybrids. The significance of these FEN1 gene localizations in human and mouse is discussed.
Amino Acid Sequence↗
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.
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Distinct roles for RAG-1 in the initiation of V(D)J recombination and in the resolution of coding ends.
Although RAG-1 and RAG-2 have been shown to be indispensible for V(D)J recombination, their exact role in this reaction remains unclear. Co-transfecting RAG-1 and RAG-2 expression vectors into NIH3T3 fibroblasts confers V(D)J recombination activity to these otherwise recombinationally inactive cells. In this report we have found that in transient transfections of mouse NIH3T3 fibroblasts with RAG-1 and RAG-2 and the appropriate recombination substrates, one RAG-1 expression vector, pRAG-1A, is capable of yielding both signal joints and coding joints, while another RAG-1 expression vector, pRAG-1B, yields only signal joints. The RAG-1 open reading frame for these two expression vectors is interchangeable, indicating that the inability to resolve coding joints is due to the 45-base pair difference found in the 5'-untranslated regions of these constructs. Differences in this region result in a 15-fold difference in gene expression when the luciferase coding region is substituted for the RAG-1 cDNA. This report provides evidence that RAG-1 may have a role in both the initiation of V(D)J recombination as well as the resolution of coding ends. The data also suggest that these RAG-1 activities may be dependent on different levels of RAG-1 expression.
3T3 Cells↗
Functional domains within FEN-1 and RAD2 define a family of structure-specific endonucleases: implications for nucleotide excision repair.
Structure-specific nucleases catalyze critical reactions in DNA replication, recombination, and repair. Recently, a structure-specific endonuclease, FEN-1, has been purified and shown to cleave DNA flap structures. Here, we describe the cloning of the murine FEN-1 gene. The nucleotide sequence of FEN-1 is highly homologous to the Saccharomyces cerevisiae genes YKL510 and RAD2. We show that YKL510 and a truncated RAD2 protein are also structure-specific endonucleases. The substrate specificity of the truncated RAD2 protein implicates branched DNA structures as important intermediates in nucleotide excision repair. The polarity of these branched DNA structures allows us to predict the placement of DNA scissions by RAD2 and RAD1/RAD10 in this reaction.
Amino Acid Sequence↗
Chimeric molecules created by gene amplification interfere with the analysis of somatic hypermutation of murine immunoglobulin genes.
We used the polymerase chain reaction (PCR) to amplify genes encoding murine immunoglobulin (Ig) lambda light-chain variable (V) regions, using DNA isolated from populations of germinal center B-cells, to study somatic hypermutation at this locus. Sequence analysis revealed that 30% of the amplified products were chimeric molecules consisting of segments of the V lambda 1 and V lambda 2 genes. Furthermore, an amplification- and cloning-associated artifact exchanged sequences between mutational variants of V lambda 1 genes. These PCR artifacts interfere with the analysis of somatic hypermutation of Ig genes. An alternative method that avoids these artifacts is suggested which involves the amplification of individual V lambda genes from single cells.
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Transcription, topoisomerases and recombination.
Transcription, DNA topoisomerases and genetic recombination are interrelated for several structural reasons. Transcription can affect DNA topology, resulting in effects on recombination. It can also affect the chromatin structure in which the DNA resides. Topoisomerases can affect DNA and/or chromatin structure influencing the recombination potential at a given site. Here we briefly review the extent to which homologous direct repeat recombination and site-specific recombination in eukaryotes are affected by transcription and topoisomerases. In some cases, transcription or the absence of topoisomerases have little or no effect on recombination. In others, they are important components in the recombinational process. The common denominator of any effects of transcription and topoisomerases on recombination is their shared role in altering DNA topology.
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The characterization of a mammalian DNA structure-specific endonuclease.
The repair of some types of DNA double-strand breaks is thought to proceed through DNA flap structure intermediates. A DNA flap is a bifurcated structure composed of double-stranded DNA and a displaced single-strand. To identify DNA flap cleaving activities in mammalian nuclear extracts, we created an assay utilizing a synthetic DNA flap substrate. This assay has allowed the first purification of a mammalian DNA structure-specific nuclease. The enzyme described here, flap endonuclease-1 (FEN-1), cleaves DNA flap strands that terminate with a 5' single-stranded end. As expected for an enzyme which functions in double-strand break repair flap resolution, FEN-1 cleavage is flap strand-specific and independent of flap strand length. Furthermore, efficient flap cleavage requires the presence of the entire flap structure. Substrates missing one strand are not cleaved by FEN-1. Other branch structures, including Holliday junctions, are also not cleaved by FEN-1. In addition to endonuclease activity, FEN-1 has a 5'-3' exonuclease activity which is specific for double-stranded DNA. The endo- and exonuclease activities of FEN-1 are discussed in the context of DNA replication, recombination and repair.
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Analysis of individual immunoglobulin lambda light chain genes amplified from single cells is inconsistent with variable region gene conversion in germinal-center B cell somatic mutation.
Responding B cells in specific immune responses diversify their immunoglobulin genes and are selected on their variant antigen receptors in the microenvironment of the germinal center. The patterns of mutations previously reported for immunglobulin (Ig) genes have supported mechanistic hypotheses of either error-prone DNA synthesis or templated variable region gene conversion as the underlying mechanism in the generation of these mutations. To assess the role of gene conversion in germinal-center somatic mutation, we chose to examine nucleotide changes in mouse lambda light chain genes which arose in response to a specific antigen. Laboratory mice possess three V lambda subexons, two of which differ from one another by only seven nucleotides, making these two subexons ideal for gene conversion. In the current study, we used six-parameter flow cytometry to isolate single lambda light chain-expressing germinal-center B cells from two different time points in a primary immune response. We then individually amplified and sequenced individual V lambda 1 genes from these single cells for mutational analysis. None of the 32 V lambda 1 genes, containing a total of 40 mutations, showed evidence of gene conversion from either of the other V lambda subexons. Features such as the replacement to silent ratio of the mutations documented at the earlier time point indicate an absence of antigen-driven selection. These data indicate that V region gene conversion does not contribute to germinal-center somatic mutation and that gene conversion is not responsible for targeting mutation specifically to rearranged Ig genes. The biological implications are discussed.
Amino Acid Sequence↗
The mechanism of V(D)J recombination: site-specificity, reaction fidelity and immunologic diversity.
Site-specific recombination reactions in higher eukaryotes are uncommon, perhaps because of the potential genomic instability that they may create. We focus this review on the issues of site-specificity, reaction fidelity and immunologic diversity in the V(D)J recombination reaction.
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V(D)J recombination in ataxia telangiectasia, Bloom's syndrome, and a DNA ligase I-associated immunodeficiency disorder.
Ataxia telangiectasia (AT) and Bloom's syndrome (BS) patients are characterized by sensitivity to radiation, increased lymphoid malignancy, and frequent translocations to the antigen receptor loci. Because of these features, there has been a persistent question as to whether the V(D)J recombinase might be abnormal in cells from these patients. Such abnormalities might be due to inappropriate to inaccurate expression of components of the V(D)J recombinase or due to mutation in a component shared between V(D)J recombination and other cellular processes, such as DNA repair. Bloom's syndrome is associated with a ligation deficiency, and this activity may contribute in the end resolution steps of both site-specific and general DNA-processing reactions. In the current study, we have activated V(D)J recombination in normal, AT, and BS fibroblasts and in fibroblasts from a patient with mutations that largely abolish DNA ligase I activity. We find that the signal and coding joint formation of the V(D)J recombination reaction are entirely normal in AT, BS, and DNA ligase I mutant cells. In addition to ruling out abnormalities of the V(D)J recombinase in AT, BS, and DNA ligase I mutant cells, these studies suggest that DNA ligase I is unlikely to be required for signal or coding end joining in the V(D)J recombination reaction.
Ataxia Telangiectasia↗
Extent to which homology can constrain coding exon junctional diversity in V(D)J recombination.
Among site-directed DNA recombination systems, V(D)J recombination is noteworthy in that identical reactants yield different recombination products at the junction of joined segments. This variation is the basis for diversity at the base of antigen receptor binding pockets and corresponds to V-(D)-J DNA junctions. An abundance of certain junctions has been noted. It has been proposed that these junctions are favoured because they occur where short regions of homology in participating coding ends might align preferentially. Here we use a system that is entirely free from cellular selection to show that the diversity of coding joints can be severely restricted when the coding ends participating in the reaction have short regions of homology. This constraint on diversity is diminished but not eliminated by terminal deoxynucleotidyl transferase, a mechanistic feature that has implications for the establishment of the immune repertoire.
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Lymphoid V(D)J recombination. Functional analysis of the spacer sequence within the recombination signal.
The V(D)J recombination reaction is directed by a pair of signal sequences, each consisting of a palindromic heptamer, an A/T-rich nonamer, and an intervening spacer sequence of 12 or 23 base pairs. The spacer sequence previously has not been analyzed for a functional role. In this study, numerous simultaneous sequence changes have been made in the spacer of each signal to test their functional importance. All of the AT base pairs in each signal were changed to GC base pairs. This particular change is of interest because it markedly increases the energy that would be required to melt out the two strands of each signal to permit the intersignal base pairing proposed in a commonly invoked model for signal-signal interaction in V(D)J recombination. We find that changing 6 to 12 AT base pairs in the 12-signal to GC does not affect V(D)J recombination, nor does changing 11 of 23 AT base pairs in the 23-signal. Substrates with all-GC spacer sequences in both the 12- and the 23-signal also recombine at efficiencies that are not significantly reduced. These studies demonstrate that the sequences at these particular positions are not recognized by the recombinase. In addition, the data do not support models invoking signal-signal base pairing.
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Coding end sequence can markedly affect the initiation of V(D)J recombination.
In V(D)J recombination, two site-specific cuts are made adjacent to V, D, and J subexons to create four DNA ends, two of which (the coding ends) are joined to generate the exon that encodes the variable domain of the antigen receptor. Although deviations from consensus signal sequences have been reported previously to have a large impact on the efficiency of V(D)J recombination, coding end sequence has been assumed to be neutral with respect to the efficiency of recombination. We have used extrachromosomal V(D)J recombination substrates to undertake a systematic comparison of coding end sequences. Substrates were constructed that contain identical consensus recombination signal sequences, where only the coding ends were varied. Surprisingly, we found that nucleotide sequence at the coding end can affect the efficiency of V(D)J recombination > 250-fold. Variable initiation of recombination appears to account for most of the effect. This finding has mechanistic implications because it indicates that signal-binding proteins involved in V(D)J recombination may have different levels of activity when confronted with coding ends of different sequence. Our results also indicate that coding end sequence must be considered to be among the major factors that shape the antigen receptor repertoire.
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