6. Molecs of Spec Recog II

Question 1

what are the 3 mechanisms of somatic diversification in humans?

Answer 1

gene rearrangement, somatic hypermutation, class switch recombination (occur in lymphocytes and non-inheritable manner)

Question 2

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?

Answer 2

variable (V), diversity (D), and joining (J) - together form the variable domain of Igs and TCR genes

Question 3

What does the V gene encode?

Answer 3

the first two complementary determining regions (CDR1 and CDR2) and the first 3 framework regions (FR1, 2, and 3)

Question 4

the D gene is only found in which genes?

Answer 4

IgH, TCRBeta, and TCRgamma

Question 5

which CDR does the D gene contribute to?

Answer 5

CDR3

Question 6

what does the J gene encode?

Answer 6

FR4

Question 7

what does the C gene encode?

Answer 7

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)

Question 8

key features of VDJ recomination?

Answer 8

” - 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.
“

Question 9

what is IgH?

Answer 9

the heavy chain

Question 10

CDR1 and CDR2 are encoded by which gene?

Answer 10

V gene (combinatorial diversity) (they match the MHC molecs)

Question 11

CDR3 is encoded by which gene?

Answer 11

V(D) J junctions (junctional diversity)

Question 12

VDJ recombination is initiated by which enzyme?

Answer 12

VDJ recombinase

Question 13

VDJ recombinase is encoded by what genes?

Answer 13

RAG-1 and RAG-2 (the recombination activating genes)

Question 14

What is the 12/23 rule?

Answer 14

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

Question 15

RAG-1 vs RAG-2?

Answer 15

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!

Question 16

what occurs in the 1st phase of gene rearrangement?

Answer 16

DNA binding to RSS, 12/23 RSS synapsis, DNA cleavage (nicking/hairpin formation) by RAG-1/-2 proteins

Question 17

what occurs in the 2nd phase of gene rearrangement?

Answer 17

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)

Question 18

what residues are the most important for RAG-1/RAG-2 binding?

Answer 18

nonamer residues (heptamer and conding end nucleotides are alos contacted but more important for cleavage than binding)

Question 19

upon binding to the RSS, the RAG proteins bring what together?

Answer 19

the two RSS (one 12mer and one 23mer RSS) together and form a synaptic complex

Question 20

what are the 2 steps of DNA cleavage in recombination?

Answer 20

1. 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)

Question 21

joining of the recombinant ends can only occur when?

Answer 21

when the RAG proteins release the ends

Question 22

_____ are usually precise fusion of the first nucleotides of the heptamers of the excised RSS

Answer 22

signal joints

Question 23

what happens to signal joints?

Answer 23

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)

Question 24

the broken, hairpin-terminated coding ends are joined rapidly by the _______ repair enzyme complex.

Answer 24

NHEJ/non homologous DNA end joining double strand DNA break repair enzymatic complex

Question 25

what make up the DNA-dependent protein kinase in recombination?

Answer 25

Ku70, Ku80, catalytic subunit (catalytic subumit is mutated in a rare human scid mutation)

Question 26

what does the DNA-PKcs (catalytic subunit) of the NHEJ repair enzyme complex do in recombination?

Answer 26

binds the broken coding ends and recruits and activates other NHEJ enzymes

Question 27

what opens up the hairpins in coding joint formation?

Answer 27

artemis

Question 28

after artemis binds the coding ends and nucleases/polymerases modify the ends (deletions and insertions), what joins them?

Answer 28

DNA ligase4 protein complex

Question 29

templated insertions in coding joint formation are called P-nucleotides and they are generated by what?

Answer 29

asymmetric opening of the hairpin ends (one strand is longer than the other, so filled in with palendromic sequence “P” by DNA polymerase)

Question 30

template-independent insertions in coding joint formation are mediated by what enzyme?

Answer 30

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

Question 31

imprecise joining results in junctional diversity - what is this due to?

Answer 31

naturally imprecise activities of NHEJ reaction, not due to the actions of the RAG enzyme

Question 32

what are the 3 levels of regulation of VDJ recombination?

Answer 32

1. 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)

Question 33

what 5 factors influence accessibility of recombinase to DNA?

Answer 33

1. 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

Question 34

accessibility hypothesis?

Answer 34

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

Question 35

Omenn syndrome

Answer 35

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

Question 36

combined scid and radiation sensitivity caused by Artemis/DNA ligase 4 mutations

Answer 36

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

Question 37

somatic hypermutation and class switch involves further somatic diversificiation of which genes?

Answer 37

Ig only

Question 38

Somatic hypermutation is point mutations in which genes?

Answer 38

IgH or IgL V genes

Question 39

class switch is recombination between which genes?

Answer 39

IgH C genes

Question 40

when/where does SHM and class switch occur?

Answer 40

mature B cells upon antigen stimulation

Question 41

what is the enzyme that initiates both SHM and class switch?

Answer 41

AID/AICDA (activation induced cytidine deaminase)

Question 42

Ig somatic hypermutation is the molecular basis for what process?

Answer 42

Ab affinity maturation

Question 43

SHM operates in most vertebrates in mature, antigen specific B-lymphocytes to increase what?

Answer 43

affinity of the already functional antibody to the target antigen

Question 44

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?

Answer 44

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

Question 45

what change is caused by AICDA?

Answer 45

C-> T transition (AICDA deaminates C residues and changes to dU which in turn behaves like T during replication)

Question 46

what 3 mechanisms allow SHM to be a much more extensive mutagenesis than the action of AICDA alone?

Answer 46

1. 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)

Question 47

what is class switch recombination?

Answer 47

the mechanism that changes the isotype of IgH chains during antigen specific response

Question 48

what is the enzymatic connection between VDJ recombination and CSR?

Answer 48

none

Question 49

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.

Answer 49

switch regions (Smu, Sgamma, Sepsilon, and Salpha)

Question 50

In the switch regoins, which are GC-rich and good targets for multiple cytidine deaminations, what type of repair dominates?

Answer 50

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)

Question 51

why is there no DNA switching between IgM and IgD?

Answer 51

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.

Question 52

the choice of isotype switching is regulated by what?

Answer 52

various stimuli and cytokines (TGFB, IFNg, IL2, IL4, IL5) ….these activate txn only at a specific switch region, making it readily targeted by AICDA

Question 53

Type 2 hyper IgM syndrome

Answer 53

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

Question 54

type 1 hyper IgM syndrome

Answer 54

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

Question 55

VDJ recombination and class swtich recombination can lead to chromosomal translocations and _____ malignancies.

Answer 55

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)