VDJ Recombination and Antibody Diversity

VDJ Recombination and Antibody Diversity

• The mammalian immune system generates an almost unlimited number of different light and heavy chain encoding genes during B cell development.
• Each B cell produces a single antibody with a unique antigen-binding site.
• Antibodies possess a variable and a constant region.
• The variable region is responsible for recognizing and binding to specific antigens.

VDJ Recombination

• VDJ recombination is a process that generates diverse antibody genes.
• DNA sequences of all stem cells are the same, but this process creates unique antibody genes encoding light and heavy chains.
• The variable region is composed of V, D, and J segments.
• During VDJ recombination, these segments are rearranged, resulting in a wide range of antibody diversity.

VDJ Recombination Steps

• VD recombination: The V and D segments are joined.
• DJ recombination: The D and J segments are joined.
• Transcription and splicing: The rearranged VDJ segment is transcribed into mRNA.

Number of VDJ Combinations

• The number of possible VDJ combinations is 150 x 9 x 4 = 5400.

VDJ Recombination and Non-Homologous End Joining (NHEJ)

• NHEJ is a DNA repair pathway that joins broken DNA ends.
• Recombinase: The RAG1 and RAG2 proteins are responsible for cutting the DNA and initiating the recombination process.
• Double-strand break (DSB) formation: The RAG proteins create DSBs with hairpin ends.
• Hairpin end removal: The Ku70/Ku80 complex and DNA-PK recruit Artemis to remove the hairpin ends.
• DSB end joining: XRCC4 and Ligase IV join the DSB ends.

Model for Homologous Recombination

• Homologous recombination is a DNA repair pathway that uses a homologous template to repair broken DNA.
• It starts with DNA resection to produce 3' single-stranded DNA (ssDNA).
• This 3' ssDNA searches for homologous sequences on the template DNA.
• DNA synthesis copies the homologous sequence from the template DNA to repair the damaged one.
• Ligation forms covalent bonds between the repaired DNA and the template, resulting in double Holliday junctions.
• Resolution separates the two joined DNA molecules, resulting in non-crossover or crossover events.

DNA Replication of the Leading Strand

• DNA replication occurs in one direction, from 5' to 3'.
• Pairing of ssDNA with the template DNA is essential for DNA extension.

DNA End Resection

• The process of producing 3' ssDNA tails from a DSB, essential for homologous recombination.
• This is achieved through nucleolytic degradation of the 5'-terminated strands, known as 5'-3' end resection.

Nucleolytic Degradation

• Cleavage of phosphodiester bonds within a DNA chain by deoxyribonuclease (DNase).
• Endonuclease: Cleaves the phosphodiester bond within a DNA chain.
• Exonuclease: Cleaves nucleotides one at a time from one end of a DNA chain.
• Digestion polarity: Can be 5' to 3' or 3' to 5' depending on the exonuclease.

Mre11 Complex

• The Mre11 complex, formed by MRE11, RAD50, and NBS1, is essential for homologous recombination.
• It binds to DSB sites at the early stages of repair.
• It possesses 3' to 5' exonuclease activity and endonuclease activity, both crucial for DNA resection.

DNA Digestion by Mre11 Complex

• The Mre11 complex uses both endonuclease and exonuclease activities to produce 3' ssDNA.
• The Sae2 protein regulates these activities, stimulating endonuclease activity but not exonuclease activity.

DNA Resection by Mre11 Complex

• The Mre11 complex initiates DNA resection by introducing a nick in the DNA, allowing the 3' to 5' exonuclease activity to create a 3' ssDNA tail.
• This pathway is more complicated than simply starting with 5' to 3' digestion because the 5' end might be blocked due to:
  • Covalently-bound proteins at 5' end.
  • Secondary structure at the end of DSB.
• The Mre11 complex-mediated resection is limited, typically 100-300 nt.

Extended DNA Resection Pathways

• Exo1: A 5' to 3' exonuclease that effectively extends the resection initiated by the Mre11 complex.
• Sgs1/Dna2:
  • Sgs1 is a DNA helicase that unwinds dsDNA to create 5'-flap ssDNA, a substrate for Dna2.
  • Dna2 is a 5'-flap endonuclease that cleaves within single-stranded regions of substrates forming flaps, thus extending the resection.

Exo1 Pathway

• Exo1 efficiently resects dsDNA with a short 3' ssDNA tail, which is the product of Mre11-mediated resection.

Sgs1/Dna2 Pathway

• Sgs1 unwinds dsDNA, creating a 5'-flap ssDNA for Dna2.
• Dna2 cleaves within single-stranded regions, extending the resection initiated by Mre11.

Explore the intricate processes of VDJ recombination and how they contribute to antibody diversity in the mammalian immune system. This quiz covers the steps involved in generating unique antibody genes and the structure of antibodies, emphasizing their importance in immune response.