6. Molecs of Spec Recog II Flashcards
what are the 3 mechanisms of somatic diversification in humans?
gene rearrangement, somatic hypermutation, class switch recombination (occur in lymphocytes and non-inheritable manner)
the variable domains of the antigen receptors is encoded by not a single, but multiple, discontinuous gene segments. The three kinds of segments are what?
variable (V), diversity (D), and joining (J) - together form the variable domain of Igs and TCR genes
What does the V gene encode?
the first two complementary determining regions (CDR1 and CDR2) and the first 3 framework regions (FR1, 2, and 3)
the D gene is only found in which genes?
IgH, TCRBeta, and TCRgamma
which CDR does the D gene contribute to?
CDR3
what does the J gene encode?
FR4
what does the C gene encode?
constant domain (has multiple exons that are spliced together through txn but does not undergo rearrangement, the first exon of the C gene is also spliced but not rearranged to the J gene during txn)
key features of VDJ recomination?
” - site specific recombination (only operates at antigen receptor loci);
- lymphoid specific (only occurs in B and T-lymphocytes, at immature stage of differentiation);
- assembles only one V, one or more D (if available) and one J genes at the DNA level;
- absolutely required for the generation of functional antigen receptors and, therefore, for lymphocyte development.
“
what is IgH?
the heavy chain
CDR1 and CDR2 are encoded by which gene?
V gene (combinatorial diversity) (they match the MHC molecs)
CDR3 is encoded by which gene?
V(D) J junctions (junctional diversity)
VDJ recombination is initiated by which enzyme?
VDJ recombinase
VDJ recombinase is encoded by what genes?
RAG-1 and RAG-2 (the recombination activating genes)
What is the 12/23 rule?
rearranging gene segments are always flanked by different length RSS (recombination signal sequence) - V gene has 23 bp and is thus a 2 turn RSS between heptamer and nonamer, while J gene has 12 bp and is thus a 1 turn RSS between heptamer and nonamer. THE HEPTAMER AND NONAMER ARE THUS ALWAYS CONTACTED ON THE SAME SIDE OF THE DNA ***this is the reason why even one-two nucleotide differences in the length of the spacer abolish binidng and cause deficiency of B and T cells
RAG-1 vs RAG-2?
RAG-1 bind the RSS directly, catalytic DDE motif, ubiquitin ligase activity; RAG-2 associates with RAG-1, increases DNA binding affinity to the RSS, facilitates binding of RAG to the chromosome; both are absolutely required for VDJ recombination and both are specific for early, immature lymphocytes!
what occurs in the 1st phase of gene rearrangement?
DNA binding to RSS, 12/23 RSS synapsis, DNA cleavage (nicking/hairpin formation) by RAG-1/-2 proteins
what occurs in the 2nd phase of gene rearrangement?
DNA broken end binding, DNA hairpin/end processing, DNA joining (coding joint/signal joint) by NHEJ proteins (non homologous end joining repair of DNA ds breaks. Ubiquitously expressed activity in ALL CELLS and NOT LYMPHOID SPECIFIC)
what residues are the most important for RAG-1/RAG-2 binding?
nonamer residues (heptamer and conding end nucleotides are alos contacted but more important for cleavage than binding)
upon binding to the RSS, the RAG proteins bring what together?
the two RSS (one 12mer and one 23mer RSS) together and form a synaptic complex
what are the 2 steps of DNA cleavage in recombination?
- nicking the top strand (precisely between the heptamer and the last nucleotide of the conding end of the gene segment) 2. hairpin formation (free 3’OH end from the top strand attacs the bottom strand and forms a hairpin on the coding end)….thus two broken DNA ends are generated: open, 5’ phosphorylated RSS ends (signal ends) and hairpin-terminated coding ends. The four ends (two at each RSS-gene segment cleavage) remain in a complex with the rag proteins for a short time (four-end complex)
joining of the recombinant ends can only occur when?
when the RAG proteins release the ends
_____ are usually precise fusion of the first nucleotides of the heptamers of the excised RSS
signal joints
what happens to signal joints?
removed from the chromosome and become extrachromosomal circles that persist in mature lymphocytes - only lost during cell division because they do not replicate, therefore VDJ recombination excision circle (REC) can be used to monitor cell division history (rarely VDJ recombination occurs with inversion and the signal joint is retained on the chromosome)
the broken, hairpin-terminated coding ends are joined rapidly by the _______ repair enzyme complex.
NHEJ/non homologous DNA end joining double strand DNA break repair enzymatic complex
what make up the DNA-dependent protein kinase in recombination?
Ku70, Ku80, catalytic subunit (catalytic subumit is mutated in a rare human scid mutation)
what does the DNA-PKcs (catalytic subunit) of the NHEJ repair enzyme complex do in recombination?
binds the broken coding ends and recruits and activates other NHEJ enzymes
what opens up the hairpins in coding joint formation?
artemis
after artemis binds the coding ends and nucleases/polymerases modify the ends (deletions and insertions), what joins them?
DNA ligase4 protein complex
templated insertions in coding joint formation are called P-nucleotides and they are generated by what?
asymmetric opening of the hairpin ends (one strand is longer than the other, so filled in with palendromic sequence “P” by DNA polymerase)
template-independent insertions in coding joint formation are mediated by what enzyme?
terminal deoxynucleotidyl-transferase (Tdt) = a lymphoid specific enzyme that can add a few N-nucleotides to the end of broken DNA molecules iwthout the need for another strand (template)….only function is to modify the rearranged gene segments
imprecise joining results in junctional diversity - what is this due to?
naturally imprecise activities of NHEJ reaction, not due to the actions of the RAG enzyme
what are the 3 levels of regulation of VDJ recombination?
- cell type and differentiation specific (only in T/B-cell precursors) 2. locus-specific (Ig rearrangement in Bcells or TCR rearrangement in Tcells) 3. allele-specific (allelic exclusion, receptor editing)
what 5 factors influence accessibility of recombinase to DNA?
- transcriptional regulatory cis-acting sequences (promotor, enhancer) 2. sequence-specific DNA binding trans-acting factors 3. transcription across unrearranged gene segments (germline txn) 4. chromatin modification (covalent and non-covalent histone modificiations) 5. subnuclear relocalization and looping of gene segments in the nucleus
accessibility hypothesis?
the nucleo-chromatin is a powerful barrier to recombination and repair so the antigen receptor locus has to become ACCESSIBLE to the recombinase. For this, RAG-2 (PHD) binds modified histone H3 to facilitate access of the VDJ recombinase to the Ig/TCR loci (re B-cells/T-cells, respectively), then RAG-1 binds the recombination signal and cleaves the DNA
Omenn syndrome
scid caused by Rag-1 (affect RSS DNA binding or catalytic activity) or Rag-2 mutations (affect chromatin accessibility of VDJ recombinase)…sxs: erythrodema, desquamation, alopecia, chronic diarrhea, FTT, lymphadenopathy, hepatosplenomegaly….lab: No B cells, oligclonal T cells (COMPLETE LACT OF B AND T LYMPHOCYTES), hyper IgE, eosinophilia…..cause: hypomorphic RAG gene mutations….treatment: bone marrow transplant and gene therapy
combined scid and radiation sensitivity caused by Artemis/DNA ligase 4 mutations
hypomorphic human artemis mutation which appears in 15% of the population causes mild radio-sensitvity and potential increased tumor susceptibility; mutations in the DNA ligase 4 cause growth retardation, microcephaly and radiation sensitivity but not significant immunodeficiency or VDJ recombination defects…sxs: neonatal scid, childhood EBV-lymphoma, adult-onset immunodeficiency, microcephaly (ligase 4), radiations sensitivity, genomic instability….lab: no T/B-cells, little or no circulating Ig….cause: complete or hypomorphic artemis or hypormorphic DNA ligase 4 gene mutations….treatment: bone marrow transplant or gene therapy
somatic hypermutation and class switch involves further somatic diversificiation of which genes?
Ig only
Somatic hypermutation is point mutations in which genes?
IgH or IgL V genes
class switch is recombination between which genes?
IgH C genes
when/where does SHM and class switch occur?
mature B cells upon antigen stimulation
what is the enzyme that initiates both SHM and class switch?
AID/AICDA (activation induced cytidine deaminase)
Ig somatic hypermutation is the molecular basis for what process?
Ab affinity maturation
SHM operates in most vertebrates in mature, antigen specific B-lymphocytes to increase what?
affinity of the already functional antibody to the target antigen
specific de novo point mutations are introduced at selected residues of already rearranged V(D)J genes of Ig heavy (IgH) and light (IgL) chain loci. This process uses a unique, B-cell specific enzyme, AICDA or AID, and what else?
error-prone DNA polymerases to mutate single nucleotides in certain positions (MUTATION HOT SPOTS), thereby changing the coding capacity of the V gene and creating a large diversity
what change is caused by AICDA?
C-> T transition (AICDA deaminates C residues and changes to dU which in turn behaves like T during replication)
what 3 mechanisms allow SHM to be a much more extensive mutagenesis than the action of AICDA alone?
- replicaiton of the dU results in C-T transition 2. removal of dU residue by UNG enzyme activates BER that can either fix the effect of AICDA or introduce any of the three other nucleotides (C-T, C-G, or C-A mutations) 3. removal of dU residues by mismatch DNA repair (MMR) apparatus and recruitment of error-prone DNA polymerases that can introduce mutations across a few nucleotides length or a few nucleotides away from the original AICDA target site (casing C-T, C-G, or C-A mutations)
what is class switch recombination?
the mechanism that changes the isotype of IgH chains during antigen specific response
what is the enzymatic connection between VDJ recombination and CSR?
none
The C genes of the IgH locus are preceded by what? These are transcribed upon B-cell activation and are particularly heavily targeted by AICDA similarly as it attacks cytidines in IgV genes.
switch regions (Smu, Sgamma, Sepsilon, and Salpha)
In the switch regoins, which are GC-rich and good targets for multiple cytidine deaminations, what type of repair dominates?
UNG-mediated BER coupled with apurinic/apyrimidinic endonuclease (APE) which creates a single strand nick in the DNA when it excises the mutant C residue…if two mutated C resides, close enough on the two strands are simultaneously repaired and nicked, it creates a dsbreak which results in excision of the intervening DNA and joining of the rest of the chromosome, deleting the C genes in betweenthem. Joining is by NHEJ)
why is there no DNA switching between IgM and IgD?
there are no switch regoins between Cmu and Cdelta…instead IgM and IgD are txn in a single mRNA and alternative splicing determines which isotype is expressed. In mature, naïve B-cells, IgM and IgD are typically co-expressed because alternative splicing is a quantitative event. In contrast, in activated B-cells, once a CSR occurred the cell can only express EXCLUSIVELY the switched isotype and cannot revert ot IgM-positive state because CSR is a permanent, qualitative change in the B-cell genome.
the choice of isotype switching is regulated by what?
various stimuli and cytokines (TGFB, IFNg, IL2, IL4, IL5) ….these activate txn only at a specific switch region, making it readily targeted by AICDA
Type 2 hyper IgM syndrome
deficiency caused by AID mutations - autosomal…AID mutations affect catalytic activity, protein trafficking or dimerization….sxs: repeated bacterial pneumonia, otitis, lymphadenopathy…lab: normal B/T cells, hyper IgM, no other Ig isotypes…cause: inactivating AID gene mutations….treatment: IV Ig…**ENLARGED GERMINAL CENTERS
type 1 hyper IgM syndrome
x-linked or auto - most common and mutations in CD40L (which is on T cells and interacts with CD40 on B cells so defective T-cell/B-cell collaboration)….similar to Type 2, but no germinal centers so no affinity maturation and isotype switch
VDJ recombination and class swtich recombination can lead to chromosomal translocations and _____ malignancies.
lymphoid (IgH/c-myc translocaiton in Non-Hodgkin Burkitt lymphoma, Bcl-6/IgH in non-hodgkin lymphoma, or Pax5/IgH in acute B-cell leukemia)
