Boyes V(D)J Flashcards
Where does VDJ recombination occur?
Heavy chain in proB cells
Light chain in preB cells
What size is each domain of an antibody?
110residues
How many polypeptides is an antibody made up of?
4
How many types of light chain constant region are there?
2- lamda and kappa
How many types of antibodies are there?
5x10^13
Which cells can rearrange the DNA?
B cells
How many combinations of segments make up a heavy chain?
40V
25D
6J
=6000
How many kappa variable segments are there?
40
How many kappa Joining segments are there?
5
How many lamda variable segments are there?
30
How many lamda joining segments are there?
4
How many combinations can VDJ create?
1.9x10^6
Which proteins bind RSS?
RAG proteins
What are the 2 types of RSS?
23bp or 12bp
What is the sequence of 23bp RSS?
heptamer-spacer-nonamer
What is the RSS sequence of kappa?
V23- 12J
What is the RSS sequence of lamda?
V12-23J
What is the RSS sequence of heavy chain?
V23-12D12-23J
What is the sequence of the 12bp RSS?
nonamer-spacer-heptamer
Is the RSS spacer sequence conserved?
No
What is the structure of RAG1?
1040 residues,
C terminal active
DDE motif
binds nonamer
What is the structure of RAG2?
527 residues
N terminal active
C terminal PHD binds 11p chromosome
binds heptamer
Do RAGs have introns?
No
What is the method of VDJ recombination?
RAGs bind RSS making synaptic complex
RAG1 produces a single strand nick
RAG2 transesterification produces a dsDNA break
NHEJC excise fragment
What is the structure of the heavy chain?
V-D-J-intron-C1-intron-Hinge-intron-c2-intron-c3
How far apart can RSS be?
20kb- 1Mb
What does RSS in the same direction cause?
Inversion
How is junctional diversity added?
Ku70:Ku80 binds ends DNA-PK:Artemis opens ends assymetrically P nucleotides added to fill in hairpin producing palindrome TdT adds N nucleotides to heavy chain exonuclease removed unpaired bases DNA ligase 4 ligates ends
Where is junctional diversity produced?
Hypervariable region 3
When is junctional diversity produced?
light chain: pre-B cell stage
Heavy chain: pro0B stage
How much variation is produced by junctional diversity?
3x10^7
Which constant chain do naive B cells express?
M or D immediately downstream
How are antibodies targetted to membranes or for secretion?
25 C terminal residues
Which antibody modifications are reversible?
Alternative splicing of IgM/D and membrane/secreted
How is affinity of IgM and IgA raised?
J protein binds C chain to make pentamers or dimers
What are the 5 isotypes of heavy chain?
M, D, E, A, G
Which heavy chain cannot be produced by class switching?
M
When does class switching occur?
In mature T cells that have been recruited to germinal centres in lymph nodes and recognising antigen.
What are switch regions?
1-10kb long G rich upstream regions
How does class switch recombination occur?
AID binds transcribed ssDNA Zn mediated C deamination UNG removes U base APE1 removes backbone to create a nick Lots of nicks, some randomly align to create dsDNA breaks NHEJC joins ends to remove fragment
Can multiple class switches occur?
Yes but must be downstream each time
What does somatic hypermutation do?
Fine tunes affinity through 3 hypervariable sites
What are the 3 mechanisms of somatic hypermutation?
AID produces G=U mismatch
1) replication produces T in half daughter cells
2) UNG removes base, lesion bypass randomly inserts base
3) MMR elements insert A-T mutations
Why does AID not produce dsDNA breaks in somatic hypermutation?
Lower concentration
What are the 4 methods of adding diversity to the variable region?
V(D)J (1.9x10^6)
Junctional (3x10^7)
Combinatorial
Somatic Hypermutation
What are the structural differences in T cell antibodies?
Only one receptor,
made of 2 chains alpha and beta
only one variable region
How is diversity added to T cell antibodies?
No somatic hypermutation, more J segments
What type of T cell antibodies are produced in the gut?
gamma and delta
How are antibodies held in membranes?
complexes using 25residue C terminal motifs
Where are antigens presented?
APC/dendritic cells with MHC2 for bacterial peptides or MHC1 for viral peptides
How do T cells recognise antigens?
MHC class 2 (+bacteria) recognised CD4 complexed antibodies on T helper cells MHC class 1(+virus) recognised by CD8 complexes on cytotoxic T cells
How is V(D)J regulated?
Limited to G1 phase by RAG2 pT490 degradation
RAGs only expressed in B and T cells
Epigenetics exclude RSS in one allele
What phase is V(D)J recombination limited to?
G1
How is V(D)J initiated?
Transcription removes H2A/B dimers to expose RSS in 35-40bp for ~6 minutes.
What does transcription of antigen receptor loci cause?
H3K4me3 which binds RAG2 PHD
Acetylation opens chromatin structure and recruits remodelling complexes through bromodomains
What are the characteristics of the enhancers of antigen receptor loci?
Tissue, stage and locus specific
How has it been proved that transcription is essential for V(D)J?
Long sterile transcripts
Insertion of terminators downstream of J61 only allows transcription downstream of J49 promoter
Enhancer KO mice do not have V(D)J
How does allelic exclusion occur in V(D)J?
Only one enhancer and only one allele in active centre of nucleus
How has allelic exclusion been proved?
2 enhancers upregulates expression
What type of disease does errors cause?
50% of all lymphiod cancers
What is the main form of error during V(D)J?
Chromosome translocation during dsDNA breaks
How can chromosome translocations occur?
cryptic RSSs End donation RAG mediated transposition ESC reintegration Cut and run
What size is the ESC?
20kb-1mb
How many mutations does RAG mediated transposition cause?
1/44,000 recombination events
Where has RAG mediated transposition occured?
THRalpha/HPT locus
IN VITRO
What protects against RAG mediated transposition in vivo?
The C regulatory domain of RAG
Where have cryptic RSSs been proved?
Sequencing of LM02 from chromosome 11 into T cell alpha locus of chromosome 14
Where have cryptic RSSs been found?
LM02/alpha
SIL/SCL
TCR/IgH
How do broken ends of chromosomes get incorporated?
RAG recognition of unusual hairpin structures
What are examples of end donation?
BCL-2/IgH
BCL-1/IgH
How common is integration of ESCs?
1/10,000 to 1/100,000 recombination events
Where do ESCs reintegrate?
Cryptic RSSs upstream of an oncogene
How do chromosome translocations cause cancer?
Bring oncogenes under control of strong promoter
What unpublished data is there for ESCs causing cancer?
Cut and run of ESC with RSS bound to RAGs producing ssDNA cuts to produce free ends
How has the link between chromosome translocation and Class switch recombination been proved?
AID KO mice can produce no Myc-IgH translocation even when stimulated with pristine and IL6 translocation stimulants
What does AID overexpression cause?
Cancer
How are chromosome translocations protected against?
Ku70:Ku80 complex hold cut ends for NHEJC to seal
