IMMUN 441 Week 4 AC Quiz Section

DNA Transcription and Translation Nucleus 5’ Germline DNA 3’ Pre-mRNA Polyadenylation Exon 1 Intron 1 Exon 2 Intron 2 Exon 3 AAAAAA Splicing AAAAAA Cytosol mRNA Open Reading Frame AAAAAA Protein

DNA Gene Rearrangement 5’ Germline DNA Gene A Gene B Gene C Gene D 3’ Permanently Lost Germline DNA Gene A Gene D

V(D)J Recombination

Structure of the BCR loci What are the proteins that carry out VDJ recombination? Which cell types are they expressed in? What would happen if a mutation caused RAG to have off target effects?

Step 1

Step 1 Step 2

Step 1 Step 2 Step 3

Step 1 Step 2 Step 3 Step 4

Step 1 Step 2 Step 3 Step 4 Step 5 Constant region of the heavy or the light chain determines the class? Step 5

-α chain variability encoded in recombined V-J segments What’s the difference between VDJ recombination in B cells versus T cells TCR Recombination: -α chain variability encoded in recombined V-J segments -β chain variability encoded in recombined D-J segments -TCR rearrangement utilizes the same proteins and mechanisms involved in Ig rearrangement

12/23 rule Why is it important to have RSS? Why can’t 23 and 23 come together or vice-versa? What would happen if you had RSS elsewhere in the genome?

Ku: DNA repair protein required for Ig and TCR gene rearrangement -forms a ring around the DNA and recruits DNA-PK

Junctional Diversity DNA-PK: recruits and activates Artemis Artemis: nuclease that nicks hairpins at a random site

Junctional Diversity TdT: inserts nontemplated nucleotides (N-nucleotides) between gene segments in V-regions

Junctional Diversity DNA ligase: enzyme that joins ends of dsDNA

N-nucleotide addition during imprecise coding joint resolution can yield junctional diversity Artemis cleaves hairpin, yielding P nucleotides V D TdT adds N nucleotides V D DNA ligase and repair joins gene segments V D Junctional Diversity This is only productive if new nucleotides are added in sets of 3 (D reading frame is preserved)

Productive vs. non-productive N-nucleotide addition during imprecise coding joint resolution: Frameshift mutations Region Amino Acid mRNA DNA (sense) GCT AAT AAC GAG CTA ATT AGC GAA AGT AAC TAT GAT GAA AGG GCU AAU AAC GAG CUA AUU AGC GAA AGU AAC UAU GAU GAA AGG A N N E L I S E S N Y D E R V D

A N N E L I S E S N Y D E R Scenario 1: Addition of 1 N-nucleotide by TdT Region Amino Acid mRNA DNA (sense) GCT AAT AAC GAG CTA ATT AGC GAA C AGT AAC TAT GAT GAA AGG GCU AAU AAC GAG CUA AUU AGC GAA AGU AAC UAU GAU GAA AGG A N N E L I S E S N Y D E R V D

A N N E L I S E Q STOP Scenario 1: Addition of 1 N-nucleotide shifts reading frame of remaining D gene, resulting in coding for truncated protein Region Amino Acid mRNA DNA (sense) GCT AAT AAC GAG CTA ATT AGC GAA CAG TAA CTA TGA TGA AAG GCU AAU AAC GAG CUA AUU AGC GAA CAG UAA CUA UGA UGA AAG A N N E L I S E Q STOP V D

A N N E L I S E S N Y D E R Scenario 2: Addition of 2 N-nucleotides by TdT Region Amino Acid mRNA DNA (sense) GCT AAT AAC GAG CTA ATT AGC GAA CG AGT AAC TAT GAT GAA AGG GCU AAU AAC GAG CUA AUU AGC GAA AGU AAC UAU GAU GAA AGG A N N E L I S E S N Y D E R V D

A N N E L I S E R V T M M K Scenario 2: Addition of 2 N-nucleotides shifts reading frame of remaining D gene, resulting in coding for non-functional protein Region Amino Acid mRNA DNA (sense) GCT AAT AAC GAG CTA ATT AGC GAA CGA GTA ACT ATG ATG AAA GCU AAU AAC GAG CUA AUU AGC GAA CGA GUA ACU AUG AUG AAG A N N E L I S E R V T M M K V D

A N N E L I S E R V T M M K Scenario 2: Addition of 2 N-nucleotides shifts reading frame of remaining D gene, resulting in coding for non-functional protein Region Amino Acid mRNA DNA (sense) GCT AAT AAC GAG CTA ATT AGC GAA CGA GTA ACT ATG ATG AAA GCU AAU AAC GAG CUA AUU AGC GAA CGA GUA ACU AUG AUG AAG A N N E L I S E R V T M M K Nonsense amino acids yield a non-functional protein domain V D

A N N E L I S E S N Y D E R Scenario 3: Addition of 3 N-nucleotides by TdT Region Amino Acid mRNA DNA (sense) GCT AAT AAC GAG CTA ATT AGC GAA CGA AGT AAC TAT GAT GAA AGG GCU AAU AAC GAG CUA AUU AGC GAA AGU AAC UAU GAU GAA AGG A N N E L I S E S N Y D E R V D

A N N E L I S E R S N Y D E R Scenario 3: Addition of 3 N-nucleotides maintains original reading frame of remaining D gene, resulting in coding for nearly identical protein Region Amino Acid mRNA DNA (sense) GCT AAT AAC GAG CTA ATT AGC GAA CGA AGT AAC TAT GAT GAA AGG GCU AAU AAC GAG CUA AUU AGC GAA CGA AGU AAC UAU GAU GAA AGG A N N E L I S E R S N Y D E R V D

A N N E L I S E R S N Y D E R Scenario 3: Addition of 3 N-nucleotides maintains original reading frame of remaining D gene, resulting in coding for nearly identical protein Region Amino Acid mRNA DNA (sense) GCT AAT AAC GAG CTA ATT AGC GAA CGA AGT AAC TAT GAT GAA AGG GCU AAU AAC GAG CUA AUU AGC GAA CGA AGU AAC UAU GAU GAA AGG A N N E L I S E R S N Y D E R New sequence diversity! (Non-germline-encoded) =Junctional diversity V D

Unique features of the BCR

Ig somatic hypermutation (SHM) and Isotype class-switching (CSR) occurs in response to B cell activation by its cognate antigen IgD IgM Sees antigen SHM Mature Naïve B cell Mature Activated B cell CSR Would these mechanisms be considered anticipatory? IgG Secretion of soluble Ig Class-switched B cell

Alternative splicing of BCR in mature naïve B cells allows coexpression of IgM and IgD isotypes Is this permanent class switching, why or why not?

Alternative splicing of BCR in activated B cells allows coexpression of transmembrane and secreted Ig

Somatic Hypermutation and Class Switch Recombination both rely on AID

Multiple potential outcomes following AID mutation Copying of the uracil Templated replication U recognized as T Opposite strand = A Transition

Multiple potential outcomes following AID mutation Copying of the uracil Removal of the uracil Templated replication U recognized as T Opposite strand = A Any base possible Nontemplated replication Does somatic hypermutation always result in higher affinity receptors? What happens to receptors with deleteriously mutations? Transition Transition or Transversion

Class switching Why does the class matter? What’s an example?

Class switching is initiated by AID targeting of switch regions

Class switching is initiated by AID targeting of switch regions

Class switching results in permanent isotype constant region excision Intronic rearrangement = always productive

Class switching results in permanent isotype constant region excision

(P- and N- nucleotides) (⅓ productive) Somatic hypermutation (SHM) Event Processes Pre- or Post-Ag? B or T cells? Nature of Change Key Enzymes DNA recombination Pre-Ag Both Irreversible Junctional diversity (P- and N- nucleotides) (⅓ productive) Somatic hypermutation (SHM) Point mutations Post-Ag B cells AID, UNG Class-switch recombination (CSR) (all productive) AID, UNG, APE-1 IgM/IgD expression Differential RNA splicing Reversible (regulated) Spliceosome Membrane bound/secreted form RAG1/2, TdT, Ku70:Ku80, DNA-PK, Artemis, DNA ligase V(D)J recombination