RESUMO
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.
Assuntos
Linfócitos B/imunologia , DNA/metabolismo , Switching de Imunoglobulina , Região de Troca de Imunoglobulinas , Hibridização de Ácido Nucleico , RNA/metabolismo , Animais , Northern Blotting , Imunoglobulinas/biossíntese , Interleucina-10/imunologia , Interleucina-4/imunologia , Lipopolissacarídeos/farmacologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Genéticos , Splicing de RNA , Recombinação Genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonuclease H/metabolismo , Ribonuclease Pancreático/metabolismo , Transcrição GênicaRESUMO
Immunoglobulin class switching is mediated by recombination between switch sequences located immediately upstream of the immunoglobulin constant heavy chain genes. Targeting of recombination to particular switch sequences is associated temporally with transcription through these regions. We recently have provided evidence for inducible and stable RNA-DNA hybrid formation at switch sequences in the mouse genome that are mechanistically important for class switching in vivo. Here, we define in vitro the precise configuration of the DNA and RNA strands within this hybrid structure at the Smicro, Sgamma3 and Sgamma2b mouse switch sequences. We find that the G-rich (non-template) DNA strand of each switch sequence is hypersensitive to probes throughout much of its length, while the C-rich (template) DNA strand is essentially resistant. These results demonstrate formation of an R-loop, whereby the G-rich RNA strand forms a stable heteroduplex with its C-rich DNA strand counterpart, and the G-rich DNA strand exists primarily in a single-stranded state. We propose that the organized structure of the R-loop is essential for targeting the class switch recombination machinery to these sequences.
Assuntos
Genes de Imunoglobulinas , Switching de Imunoglobulina , Transcrição Gênica , Animais , Sequência de Bases , DNA/química , DNA/genética , DNA Topoisomerases Tipo II/genética , Camundongos , Dados de Sequência Molecular , RNA/química , RNA/genética , Recombinação GenéticaRESUMO
Homologous genetic recombination is an essential biological process that involves the pairing and exchange of DNA between two homologous chromosomes or DNA molecules. It is of fundamental importance to the preservation of genomic integrity, the production of genetic diversity, and the proper segregation of chromosomes. In Escherichia coli, the RecA protein is essential to recombination, and biochemical analysis demonstrates that it is responsible for the crucial steps of homologous pairing and DNA strand exchange. The presence of RecA-like proteins, or their functional equivalents, in bacteriophage, other eubacteria, archaea, and eukaryotes, confirms that the mechanism of homologous pairing and DNA strand exchange is conserved throughout all forms of life. This review focuses on the biochemical and physical characteristics of DNA strand exchange proteins from three diverse organisms: RecA protein from E. coli, UvsX protein from Bacteriophage T4, and RAD51 protein from Saccharomyces cerevisiae.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Proteínas de Membrana/fisiologia , Recombinases Rec A/metabolismo , Recombinação Genética , Proteínas Virais/fisiologia , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Bacteriófago T4/genética , DNA/metabolismo , DNA/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Hidrólise , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Conformação Proteica , Rad51 Recombinase , Recombinases Rec A/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
The Rad51 protein of Saccharomyces cerevisiae is a eukaryotic homolog of the RecA protein, the prototypic DNA strand-exchange protein of Escherichia coli. RAD51 gene function is required for efficient genetic recombination and for DNA double-strand break repair. Recently, we demonstrated that RecA protein has a preferential affinity for GT-rich DNA sequences-several of which exhibit enhanced RecA protein-promoted homologous pairing activity. The fundamental similarity between the RecA and Rad51 proteins suggests that Rad51 might display an analogous bias. Using in vitro selection, here we show that the yeast Rad51 protein shares the same preference for GT-rich sequences as its prokaryotic counterpart. This bias is also manifest as an increased ability of Rad51 protein to promote the invasion of supercoiled DNA by homologous GT-rich single-stranded DNA, an activity not previously described for the eukaryotic pairing protein. We propose that the preferred utilization of GT-rich sequences is a conserved feature among all homologs of RecA protein, and that GT-rich regions are loci for increased genetic exchange in both prokaryotes and eukaryotes.
Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Sequência de Bases , DNA/química , Proteínas de Ligação a DNA/química , Dados de Sequência Molecular , Nucleotídeos/química , Nucleotídeos/metabolismo , Rad51 Recombinase , Recombinases Rec A/química , Recombinases Rec A/metabolismo , Recombinação Genética , Homologia de Sequência de AminoácidosRESUMO
The RecA protein and other DNA strand exchange proteins are characterized by their ability to bind and pair DNA in a sequence-independent manner. In vitro selection experiments demonstrate, unexpectedly, that RecA protein has a preferential affinity for DNA sequences rich in GT composition. Such GT-rich sequences are present in loci that display increased recombinational activity in both eukaryotes and prokaryotes, including the Escherichia coli recombination hotspot, chi (5'-GCTGGTGG-3'). Interestingly, these selected sequences, or chi-containing substrates, display both an enhanced rate and extent of homologous pairing in RecA protein-dependent homologous pairing reactions. Thus, the binding and pairing of DNA by RecA protein is composition-dependent, suggesting that a component of the elevated recombinational activity of chi and increased genomic rearrangements at certain DNA sequences in eukaryotes is contributed by enhanced DNA pairing activity.