BIOC2306 VDJ recomb

Domains of an antibody

antigen binding domains (NH2 domains) made from VL and VH

Both heavy chains form Fc region

heavy chains linked by disulfide bridge in hinge region. L to H chain by s-s too

V and C regions

what are the hypervariable regions

generagte high level of antigen specificity (complementarity determining region)

specific regions within variable domains of H and L chain - each have 3

loops that form antigen bindning site

3rd hyp variable region most variable

what gene segments make up the variable region

VDJ

number of gene segments for kappa light chain

40, 0, 5

number of gene segments for lambda light chain

30, 0, 4

number of gene segments for heavy chains

40, 25, 6

does recombination take place unidirectionally and what is the rule

yes

RSS with 12 and 23 bp spacers can only recombine

structure of RSS that flank the gene segments (upstream or downstream of it, unidirectional recombination)

heptamer-12/23 spacer- nonamer

what recognises RSS’s

RAG Proteins

RAG1- makes nick in strand

RAG2- binds chromatin

RAG1 vs RAG2

RAG1- c-terminal core is active region, with DDE motif in active site

RAG2- N terminal core is active region, with PHD finger req for chromatin binding

overview of VDJ recomb

RAG proteins bind RSS, bring together to form synaptic complex

RAG1 cause nick on 1 strand- resulting 3’OH attacks opposite strand in direct transesterification reaction - forming hairpin structure at coding ends and blunt dsDNA breaks at signal end

each get processed different

what binds the ends made by RAG proteins

Ku70:Cu80

tyep of reaction where 3’OH invades other strand after RAG cleavage

direct transesterification reaction

how are coding joints processed

DNA-PK:Artemis recruited by Ku70:Ku80, which open hairpin

TdT processes DNA ends (terminal deoxyucleotidyl transferase)

DNA ligaseIV:XRCC4 ligates DNA ends

how are signal joints processed

Ku70:Ku80 binds, DNA ligase:XRCC4 ligate DNA ends together

how are P and N nucleotides introduced

asymmetric opening of hairpin by artemis to generate P nucleotides (palindromic) P nucleotides from each coding joint,

TdT adds nontemplated N nts onto end of P nucleotides

segment strands pair, exonuclease removes unpaired nucleotides,

synth nts in gaps and ligate

what cells express TdT

only in Pro-Bcells and early T cells

what hypervariable region are P and N nts added

hypervariable region 3

all methods of creating antigen receptor diversity

combinatorial joining of gene segments

junctional diversification (N an P nts) when join gene segments

combinatorial joining of H and L chains

what causes transcription after recombination (VDJ)

V gene promoter activated by a strong B cell sepecific enhancer (downstream) that has now been brought closer due to gene segement removal

enhancer within the intron of C gene segments

are different domains of the constant region coded by different exons

yes

are N nucleotides present in heavy and light chains equally

no, present more in heavy chains

as TdT only exp in early stages of recomb, when heavy chain is rearranged, but not after when light chain is rearranged

stages of B cell development

pro-B cell synth heavy chain

Pre - present heavy chain Ab with surrogate light chain

immature B cell then synth light chain which it can present

all in the bone marrow

then mature in the secondary lymphoid organs

what heavy chain gene segment doesnt have a RSS uptream of it

delta heavy chain (IgD), expressed by alt. splicing (get differential expression)

how are ig either presented or secreted

dep if c terminal bit added or not, for membrane anchorage

what ig’s activate complement

igM and igG

what is the predominant ig in blood

IgG

how do different ig isotypes differ

in their ability to activate complement and their interactions with Fc receptors

ability to interact with other immune cells/activate them

location too

class switch recomination

switch to diff isotypes of ig

excise circular DNA

are IgM and IgD expressed by naive B cells by differential expression

yes

does class switch recomb occur in activated B cells

yes, swtich the class of the constant region

does class switching need to be activated by transcription

yes, thru switch sequences. making it transiently single stranded to allow AID to bind

function of AID

bind ssDNA

cat deamination of cytidine (prod uracil) therefore GU mismatch

this modification occurs in switch regions

what recognises a GU mismatch

uracil DNA glycosylase, removes uracil base (form abasic/apyrimidinic site)

APE1 (apurinic/apyrimidinic endonuclease) excises ribose to form a single strand nick in the DNA

Req occur twice to form dsDNA break

what follows the introduction of dsDNA break in switch regionsn

DNA-PK and other repair proteins act to initiate ds break repair (dsbr)

the dsbr machinery joiun the two switch regions and excises teh intervening sequences. Now new constant region downstream of VDJ

where does hypermutation primarily create diversity

in hypervariable regions

to increase antibody diversity

what triggers somatic hypermutation

AID and uracil DNA glycosylase

3 causes of hypermutation

TLS polymerase insert bases at random opposite abasic site

replication over the mismatch causes G to A transition

recruit mismatch repair machinery that cut out around mismatch and insert mutations by accident (by pol) (muts perferentially at A:T)

what are the only reversible changes in B cells

IgM and IgD expression on surface (alt. splicing)

expression of memb bound and secreted form

does switch recombination and somatic hypermutation occur in T cells

NO, AID not expressed

what are similar features of the B cell and T cell antigen receptor gene assembly processes

both assemble v region (VDJ recomb)

both have junctional diversity

transcription is activated by bringing enhancer in close proximity to promoter

do membrane bound antibodies form complexes

yes

Do T cell receptors associate with a signalling complex

yes, so can signal in cytosol when bound

how many antigen binding sites does a T cell receptor have

one ,

T cell receptor formed by alpha and beta chain

what does teh T cell receptor recognise

the antigen and the MHC protein

(with costim mols causes naive T cell activation)

what undergoes VDJ recomb to make the T cell receptor

the alpha and beta chain locus

follow the 12/23 rule too

do the alpha and beta chains of teh T cell receptor have D segments

alpha doesnt

beta does

are T cell receptors always memebrane bound

yes

what contributes most to the diversity of T cell receptors

most in hypervariable region 3,

generated by junctional diverity (P and N nts) and the larger number of J gene segemtns

what restricts VDJ recomb to G1 phase

cell-cycle dependent phosphorylation of RAG2 (binds the RSS)

pi rag2 in G1/M trasnition, causing Ub med degredation (pi T490)are the

so while RAG1 levels are constant, RAG2 levels only increaes in G1, but gets degraded otherwise. So VDJ recomb in G1 only

are the RSSs that RAG1/2 recognise in B and T cells the same

yes

what heavy chain gets rearranged in early B cell developmenet

meu heavy chain

is the first one expressed

prod the others by alt splicing or class switching

what chain gets rearranged in T cells first

beta chain

then alpha later

what mechansim restricts expression to one allele per cell

allelic exclusion

1 heavy chain recomb first, then if viable recombine one light chain (replaces surrogate light chain)

kappa light chain more freq used

how is RAG cutting regulated

differential accessiblity of RSSs

only expose correct RSS at correct development stage

usually only make RSSs more available on one of the two alleles