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Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
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The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...
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Organisms are capable of detecting and fixing nucleotide mismatches that occur during DNA replication. This sophisticated process requires identifying the new strand and replacing the erroneous bases with correct nucleotides. Mismatch repair is coordinated by many proteins in both prokaryotes and eukaryotes.
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Crossing over is the exchange of genetic information between homologous chromosomes during prophase I of meiosis I. Genetic recombination gives rise to allelic diversity in the newly formed daughter cells. In humans, crossing over produces genetically distinct haploid egg and sperm cells that undergo fertilization to produce unique offspring. Before cell division starts, the germ cell’s chromosome(s) undergo duplication in the S phase of the cell cycle. As the cells enter prophase I,...
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DNA replication is initiated at sites containing predefined DNA sequences known as origins of replication. DNA is unwound at these sites by the minichromosome maintenance (MCM) helicase and other factors such as Cdc45 and the associated GINS complex.The unwound single strands are protected by replication protein A (RPA) until DNA polymerase starts synthesizing DNA at the 5’ end of the strand in the same direction as the replication fork. To prevent the replication fork from falling apart,...
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Other than maintaining genome stability via DNA repair, homologous recombination plays an important role in diversifying the genome. In fact, the recombination of sequences forms the molecular basis of genomic evolution. Random and non-random permutations of genomic sequences create a library of new amalgamated sequences. These newly formed genomes can determine the fitness and survival of cells. In bacteria, homologous and non-homologous types of recombination lead to the evolution of new...
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Related Experiment Video

Updated: Jun 21, 2025

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae
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RNA 2'-O-methylation promotes persistent R-loop formation and AID-mediated IgH class switch recombination.

Muzaffer Ahmad Kassab1,2, Yibin Chen3,4, Xin Wang3,5

  • 1Department of Cancer Genetics and Epigenetics, Beckman Research Institute, City of Hope Medical Center, Duarte, CA, 91010, USA. muzaffer.kassab@pennmedicine.upenn.edu.

BMC Biology
|July 8, 2024
PubMed
Summary
This summary is machine-generated.

Ribose 2'-O-methylation (2'-OMe) stabilizes RNA-DNA hybrids (R-loops) at the immunoglobulin heavy chain locus, promoting immunoglobulin class switch recombination (CSR). This modification is facilitated by fibrillarin (FBL) interacting with activation-induced cytidine deaminase (AID) and snoRNA aSNORD1C.

Keywords:
2'-O-methylation (2'-OMe)Activation-induced cytidine deaminase (AID)Class switching recombination (CSR)R-loop

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Analysis of Somatic Hypermutation in the JH4 intron of Germinal Center B cells from Mouse Peyer's Patches
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Area of Science:

  • Molecular Biology
  • Immunology
  • Genetics

Background:

  • RNA-DNA hybrids (R-loops) have dual roles in genomic instability and immunoglobulin class switch recombination (CSR).
  • The mechanisms governing R-loop functions and their protection during CSR are not fully understood.
  • The persistence of CSR-associated R-loops, resistant to RNase H, remains elusive.

Purpose of the Study:

  • To investigate the regulatory mechanisms of R-loop stability during immunoglobulin class switch recombination (CSR).
  • To elucidate how R-loops contribute to both genomic instability and protective CSR.
  • To identify factors involved in the modification and persistence of R-loops at the immunoglobulin heavy chain locus.

Main Methods:

  • Analysis of R-loop modification during CSR.
  • Investigation of protein-RNA interactions involving fibrillarin (FBL), activation-induced cytidine deaminase (AID), and snoRNA aSNORD1C.
  • Gene expression disruption studies (FBL, AID, aSNORD1C) and assessment of R-loop stability and CSR.
  • Assessment of AID targeting to the immunoglobulin heavy chain locus.

Main Results:

  • R-loops at the immunoglobulin heavy (IgH) chain locus are modified by ribose 2 -O-methylation (2 -OMe) during CSR.
  • Fibrillarin (FBL) interacts with activation-induced cytidine deaminase (AID) and snoRNA aSNORD1C to mediate 2 -OMe.
  • Disruption of FBL, AID, or aSNORD1C impairs 2 -OMe, R-loop stability, and CSR.
  • FBL, AID, and aSNORD1C promote AID targeting to the IgH locus.

Conclusions:

  • Ribose 2 -O-methylation (2 -OMe) stabilizes IgH-associated R-loops, enabling productive CSR.
  • This study reveals a novel mechanism for regulating R-loop stability during CSR.
  • Findings shed light on AID-mediated CSR and the dual role of R-loops in genomic stability.