Transcript V(D)J Recombination Errors

Immunoglobulin Gene
Rearrangement
Alison Brittain
Mahboubeh Noori (Noora)
Christian Showalter
MCB 7200
Dr. Horodyski
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Outline:
• Introduction to immunoglobulins and V(D)J
recombination
• Mechanism of V(D)J recombination
• Errors in V(D)J recombination
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Immunoglobulins (Ig’s):
• Secreted by B cells and act as B cell receptors (BCR)
• Binds antigens = any substance eliciting an immune
response
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Role of Immunoglobulins (Ig’s) in Antigen
Recognition:
• Appropriate Ig’s are secreted by B cells after they differentiate
into plasma cells in the germinal center of a lymph node
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Ig Isotypes:
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Ig Structure and Variability in Antigen
Recognition – V(D)J Segments:
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V(D)J Recombination:
• 44 variable, 27 diversity, 6 joining
• 3×1011 possible combinations
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Rearrangement of Ig gene segments:
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http://what-when-how.com/wp-content/uploads/2012/04/tmp4B108.jpg
How Does Rearrangement Occur?
• Rearrangement occurs between specific sites on the
DNA called Recombination Signal Sequences (RSSs).
• Rearrangement is catalyzed by two Recombination
Activating Genes: RAG-1 and RAG-2.
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Recombination Signal Sequence (RSS):
• A short DNA sequence containing a conserved heptamer and nonamer
separated by either 12 or 23 base pair, indicate the sites of recombination
• Two types of RSS exist
• Each V, D, or J gene segment is flanked by RSS
• The RSSs are present on the 3’ side (downstream) of a V region and the
5’ side (upstream) of the J region
V, D, or j coding flank
Consensus 12 or 23 -RSS
12 or 23
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Schatz, David G., and Patrick C. Swanson. "V (D) J recombination: mechanisms of initiation." Annual review of genetics 45 (2011): 167-202.
Recombination Activating Gene (RAG):
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Schatz, David G., and Patrick C. Swanson. "V (D) J recombination: mechanisms of initiation." Annual review of genetics 45 (2011): 167-202.
V(D)J recombination:
Nicking: first step of DNA cleavage by RAG in which
one DNA strand is broken 5’ of the heptamer
Hairpin formation: second step of DNA cleavage by
RAG in which the 3’-hydroxyl of the nicked strand
attacks the other strand
HMGB: high mobility group box
Paired (synaptic) complex (PC): protein-DNA complex
in which the two RSSs are held in close juxtaposition by
the RAG proteins
Signal end: after DNA cleavage by the RAG proteins,
the DNA end that terminates in the RSS
Coding end: after DNA cleavage by the RAG proteins,
the DNA end that terminates in the coding segment
CSC: cleaved signal complex
SEC: signal end complex
Nonhomologous end joining (NHEJ): a DNA repair
process that joins broken DNA ends (double-strand
breaks) without using homologous DNA as a template
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Schatz, David G., and Patrick C. Swanson. "V (D) J recombination: mechanisms of initiation." Annual review of genetics 45 (2011): 167-202.
V(D)J recombination:
RSS
Rag-1 + Rag-2
DNA-PK
Artemis
Ku
DNA ligase/XRCC4 dimer
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https://www.youtube.com/watch?v=QTOBSFJWogE
Products of V(D)J recombination:
RSSs relative orientation determines whether
the reaction proceeds by inversion or by
deletion
Inappropriate joining of a
coding end to an RSS
Roth, David B. "V (D) J Recombination: Mechanism, Errors, and Fidelity."Microbiology Spectrum 2.6 (2014).
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V(D)J Recombination Errors:
• The rearranging of genes to generate the large
amount of antigen-receptor diversity is prone to
errors.
• Deleterious genomic rearrangements can be
potentially created as a result of V(D)J
recombination.
• Chromosome translocations can result in
cancerous growths, such as in lymphomas and
leukemia, as a result of kilo base to mega base
inversions and deletions.
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V(D)J Recombination Errors:
• There are two main categories of errors as a result
of V(D)J recombination
1. Errors in Target Recognition
2. Errors in End Joining
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Errors in Target Recognition:
• When errors in V(D)J recombination occur in
lymphoid neoplasms it may result in deleterious
chromosomal rearrangements
• First type of error is due to when an RSS and a
DNA sequence resembling RSS known as
“cryptic RSS” are recognized
• Small size of RSS sequences and the fact that
recombination does not necessitate strict
adherence of consensus heptane/monomer
sequences allows for cRSSs capable of supporting
recombination about once per kilobase in random
DNA sequences
•
Roth D. 2014. V(d)j recombination: mechanism, errors, and
fidelity. microbiolspec 2(6):
doi:10.1128/microbiolspec.MDNA3-0041-2014
Analysis of human lymphoid neoplasms have
revealed chromosome translocations involving
mismatching of RSSs and cRSSs that are adjacent
to proto-oncogenes
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Errors in Target Recognition Continued:
• Errors in mismatched RSSs and
cRSSs can cause inappropriate
expression of a target gene due to
presence of a transcriptional regulator
on the antigen-receptor loci
• Pairs
of
cRSSs
can
create
chromosome translocations such as in
T-cell acute lymphoblastic leukemia
cases
involving
translocations
between T-cell receptor gene
segments and the SCL locus in trans
• Pairs
of
cRSSs
can
create
chromosome translocation events in
cis by generating a deleted coding
joint and an excised signal joint
Roth D. 2014. V(d)j recombination: mechanism, errors, and
fidelity. microbiolspec 2(6):
doi:10.1128/microbiolspec.MDNA3-0041-2014
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Errors in Joining:
• Errors in joining involve events that join
a RAG-mediated double-strand break to
a broken DNA end created by a nonRAG-mediated mechanism
• A pair of breaks created during normal
V(D)J recombination are mistakenly
joined to another break created by
another mechanism
• Chromosome translocations or
insertions of signal-ended fragments
into another chromosomal location can
occur
Roth D. 2014. V(d)j recombination: mechanism, errors, and fidelity. microbiolspec 2(6): doi:10.1128/microbiolspec.MDNA3-0041-2014
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Chromosomal Rearrangements Cause
B- and T-cell Neoplasms:
• Neoplasms are new and abnormal
growths of tissue on the body
• Lymphomas are malignant
growths of lymphatic tissue where
lymphocytes are produced
• A Chromosomal rearrangement as
a result of V(D)J recombination
can lead to may types of nonHodgkin lymphomas such as
follicular lymphoma, Burkitt’s
lymphoma, and Chronic
Myelogenous Leukemia
Blausen.com staff. "Blausen gallery 2014". Wikiversity Journal of
Medicine. DOI:10.15347/wjm/2014.010. ISSN 20018762.
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Diseases Associated with Errors in
V(D)J Recombination:
• Burkitt’s lymphoma is a cancer of mostly B
lymphocytes. C-myc is moved from its usual
position on chromosome 8 to a location close
to enhancers of the antibody heavy chain
genes on chromosome 14 and its transcription
is significantly increased. C-myc is a
transcription factor playing a role in mitosis
of mammalian cells which produces
cancerous lymphocytes.
• CML is a form of leukemia characterized by
the increased and upregulated growth of
predominately myeloid cells in the bone
marrow and accumulation of them in the
blood as a result of constitutive tyrosine
kinase activity that activates a cascade of
proteins that control the cell cycle and speeds
up cell division. The BCR-ABL protein is
also known to inhibit DNA repair
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Regulation of V(D)J Recombination:
• Recombinase is active only in the appropriate lymphocytes
at specific times in development
• RAG1 and RAG2 are carefully limited in cells and in stages
of development
• Another control is cell-cycle-specific protein degradation of
the RAG2 protein mediated by threonine phosphorylation
(T490)
• Autoubiquitylation of RAG1 may also play a role in
regulating V(D)J recombination and preventing errors
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Summary
• V(D)J Recombination is a very useful mechanism to
create an almost limitless supply of different
antibodies to target antigens associated with
pathogens, however, this mechanism can have
errors associated with it as a result of the double
stranded breaks in DNA which may sometimes lead
to cancerous cells
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References
• Schatz, David G., and Patrick C. Swanson. "V (D) J recombination:
mechanisms of initiation." Annual review of genetics 45 (2011): 167-202
• https://www.youtube.com/watch?v=QTOBSFJWogE
• http://what-when-how.com/wp-content/uploads/2012/04/tmp4B108.jpg
• Roth D. 2014. V(d)j recombination: mechanism, errors, and fidelity.
microbiolspec 2(6): doi:10.1128/microbiolspec.MDNA3-0041-2014
• Blausen.com staff. "Blausen gallery 2014". Wikiversity Journal of
Medicine. DOI:10.15347/wjm/2014.010. ISSN 20018762.
• http://image.slidesharecdn.com/neoplasia-robbinspath-120704091051phpapp02/95/neoplasia-robbins-path-33-728.jpg?cb=1341393120
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