Recombination, Phase Variation and Antigenic Variation

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• Recombination, Phase Variation and Antigenic
  Variation

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Why Recombination?
Mutation happens - without recombination,
mutation target would increase from gene to
entire chromosome Recombination allows favorable
and unfavorable mutations to be separated
Provides a means of escape, to generate new
combinations of genes, and spreading of favorable
alleles
Two Broad Categories Homologous (or general)
Site-specific (e.g. phage genomes into bacterial
chromosomes)
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Homologous Recombination
Required for DNA replication, repairs accidents
at replication fork Repairs double strand DNA
(dsDNA) breaks Occurs at meiosis
(cross-overs) Happens at four strand stage of
meiosis, involves two of four strands Occurs
randomly between homologous sequences Double
strand break repair (DSBR) model Allelic and
non-allelic (ectopic recombination)
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General Recombination often involves a Holliday
Junction

• Proposed by Dr. Holliday (Holliday R. 1964. A
  mechanisms for gene conversion in fungi.
  Genet. Res. 5282-304)
• Recombination intermediate, also called
  cross-strand exchange
• Between two pairs of strands, one crossing and
  one non-crossing
• Can resolve in two ways depending on second DNA
  cut

Patch recombinant - Original pair of crossing
strands cut
Two recombinant chromosomes - Opposite pair of
non-crossing strands cut
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Holliday structures (part I)
Sister chromotids
e.g. 4 strand during meiosis
Sister chromotids
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Holliday structures (part II)
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Recombination is initiated by double-strand
breaks in DNA
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Mechanisms of Gene Conversion/Recombination

• Double-strand break (DSB) repair
• Synthesis-dependent strand annealing (SDSA)

Chen J et al. Nature Reviews Genetics. 2007.
8762-775
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Double-stranded (dsDNA) breaks are not uncommon
Meiosis Created by topoisomerase-like
enzymes Mitosis Radiation Mutagens (e.g.
chemicals) Stalled replication forks Specialized
endonucleases (eg site-specific HO endonuclease
in switching of yeast matting type (MAT) genes)
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Recombination requires DNA binding proteins

• Extensively studied in model organisms, E. coli
  and yeast
• Bacterial recombination enzymes identified by Rec
  - mutations
• At least 25 proteins are involved in homologous
  recombination in E. coli
• Remember four RecBCD and RecA

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RecBCD
3 member protein complex with endonuclease
and helicase activity essential for 99 of
recombination events occurring at
double-stranded breaks in bacteria binds double
stranded break unwinds and degrades
DNA Pauses at chi sequence Loads RecA on 3
ssDNA extensions
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Initiation of recombination by the RecBCD enzyme
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RecA enables single stranded DNA to invade DNA
helix

• RecA
• Involved in SOS response required for nearly
  ALL homologous
• recombination in bacteria
• Single-strand DNA binding protein, DNA dependent
  ATPase
• Multiple DNA binding sites
• Initiates the exchange of DNA between two
  recombining DNA double helixes

Eukaryotes have multiple homologs of bacterial
RecA (Rad51 is best studied)
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Chi site ?
Recombination hotspot Modifies RecBCD enzymatic
activity 5 GCTGGTGG 3 1009 chi (?) sites in
E. coli genome ? homologs in other bacteria
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Kowalczykowski TIBS. 2000. 25 156-65
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Targeted gene disruption by homologous
recombination
Lodish et al. Molecular Cell Biology
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Gene Conversion
A special type of homologous recombination Non-re
ciprocal transfer of genetic material from a
donor sequence to a highly homologous
acceptor sequence Initiated by double strand
DNA (dsDNA) breaks 5 gt 3 exonucleases 3
ssDNA tail strand invasion (RAD51 and
others) Outcome portion of donor sequence
copied to acceptor and original donor copy
unchanged
gene conversion
donor
acceptor
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Gene Conversion is not uncommon
Yeast mating type switch (MAT) genes Human
repetitive sequence elements (Alu and LINE-1
sequences) Human gene families (e.g. MHC
alleles, Rh blood group antigens,
olfactory receptor
genes) Chicken B cells Ig gene
diversification Pathogen clonal antigenic
variation (e.g. African Trypanosomes
and
Babesia bovis)
Chen et al. 2007 Gene conversion mechanisms,
evolution and human disease Nature Reviews
Genetics. 8 762-775.
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Clonal Antigenic Variation in Pathogenic Protozoa
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Two Pathogens Two Approaches
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Common Themes of Clonally Variant Antigens
Large families of non-allelic genes (one gene ON,
others OFF) Antigens are highly immunogenic but
poorly cross-reactive Switching occurs at high
but variable rate Switching is frequently
accomplished by duplicative gene conversion into
an expression site or DNA rearrangement Recombina
tion generates diversity in gene families Means
for survival and transmission
Kyes S. et al. Annu Rev. Microbiol. 2001.
55673-707
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Variations on a Theme
African Trypanosomes 1000s of VSG gene/gene
fragments dedicated expression
site recombination mediated switching
(RAD51-associated) Plasmodium falciparum 60
var genes in situ expression (no dedicated
expression site) primarily non-recombinational
switching
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Clonal Antigenic Variation in Trypanosoma brucei
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Trypanosome antigen switching
At each wave, different VSGs are expressed Switch
rates - 10-2 to 10-6 per cell in blood gt100 VSGs
expressed sequentially in one rabbit Switch not
induced by the immune system Semi-programmed --
early VSGs are always early
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Variant surface glycoproteins
Completely cover the blood-stage tryp in a tight
coat (107 /cell) except the flagellar
pocket Glycolipid anchor (released by
phospholipase C) VSG protein -- 450 amino
acids C-term is more conserved (not
exposed) N-term highly variable sequence
3-D structures are very similar
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VSG Proteins Have Diverse Sequence but Related
Structure

• The crystal structure was compared between two
  VSGs
• Despite low sequence similarity the structures
  were remarkably similar
• Conclusion Antigenic variation in trypanosomes
  is accomplished by sequence variation and not by
  gross structural alteration.

Blum M et al. Nature. 1993. 362 603-9
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VSG Expression Occurs From Unique Telomeric
Expression Sites
a-amanitin resistant Pol I promoter
Telomeric repeats
70-bp repeat
ESAGs
VSG
7
6
5
4
8
3
2
1

• VSG are expressed in long polycistronic messages
  (gt40kb)
• Expression site encodes multiple expression site
  associated genes
• There are approximately 20 bloodstream expression
  sites, only one is
• active at a time
• There are two distinct types of expression sites
• Bloodstream (above)
• metacyclic

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One VSG Expression Site is Active at a Time
70-bp repeat
ESAGs
VSG
Active Site (one)
Full-length transcript, high level
70-bp repeat
ESAGs
VSG
Inactive silent site (many)
Partial transcripts, low level
Unable to force two expression sites to be
simultaneously active Chaves et al 1999. EMBO J
184846-55
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Active VSG is Located in Subnuclear Compartment
Expression Site Body
Nucleolus and Expression Site Body
(ESB) Labeled by PolI antibody
ESB
ESB
Nucleolus
Active VSG locus tagged with Lac operator
and Visualized with Tagged lac Repressor LacI-GFP
Navarro M Gull K. 2001. Nature 414759-763.
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Three Distinct Mechanisms of VSG switching
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VSG Genome Organization
Location VSG
Silent subtelomeric VSG arrays
Telomeric VSGs
VSGs in bloodstream expression sites
Megachromosomes and intermediate chromosomes
Minichromosomes 50-100kb
1250-1400
150-250
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Size VSG pool
Taylor Rudenko. Trends Genet 2006. 22614-20
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vsg Gene Diversification
Gene conversion is likely to be a primary
mechanism to generate vsg gene diversity Most vsg
are pseudogenes Limited number of functional
genes, 7 of 806 vsg genes A reservoir of
potential genetic change contained in
non-functional vsg pseudogenes
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VSG Switching by Gene Conversion Frequently
Relies on Homology Upstream and Within the 3
Conserved Region of Genes
3conserved region
70-bp repeat
B
A
Silent VSG array
Active VSG expression site
70-bp repeat arrays
C
Switch
B
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Clonal Antigenic Variation in Plasmodium
falciparum
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Plasmodium falciparum Antigenic Variation and
Cytoadhesion are Linked
PfEMP1 switch, binding/antigenicity changes
Miller 2002
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The A,B,Cs of var Organization
60 genes per parasite haplotype (few
pseudogenes) One gene on, the others
off Similar A,B,C gene organization between
parasite isolates As are not under strong CD36
selection, others are
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PfEMP1 proteins have multiple receptor-like
domains
60 proteins Different protein forms
Common adhesion trait CD36 (blood vessels, immune
cells) Rarer ICAM-1 (blood vessels, immune
cells) Rosetting with uninfected
erythrocytes Pregnancy restricted CSA
Binding determines IE tropism
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Multiple Layers of Gene Control
Layer 1 Gene structure and putative regulatory
elements
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Var Genes are Expressed in situ
Sterile Transcripts
DBL
CIDR
DBL
DBL
ATS
TM
Exon 2 Conserved
Exon 1 hypervariable

• Monocistronic
• Only one var gene is expressed at a time
• No dedicated expression sites, genes are
  expressed in situ
• Transcription factors? Members of ApiAP2 family?

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var Intron Promoter May Cooperate in Gene
Silencing
Default var promoter state
Active
luciferase
var upstream region
promoter pairing
luciferase
Silent
intron promoter
var upstream region
luciferase
Active
disabled intron promoter
var upstream region
Deitsch et al. Nature. 2001. 412875-6
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• Note others argue var promoter is sufficient to
  silence genes
• Voss et al. (2006) A var promoter controls
  allelic exclusion of virulence genes in
  Plasmodium falciparum malaria

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Layer 2 Chromatin modifications
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Silent and Active Chromatin Marks
Note similar marks are found at many active
and silent genes
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SIR2 Regulates the Silencing of Some Var Genes

• Silent information regulator (SIR) proteins
• associate with the ends of chromosomes in
• yeast and Plasmodium.
• Deacetylation of histones by SIR2 can initiate
• the establishment of heterochromatin (silent
• chromatin in which transcription is
• repressed).
• silent subtelomeric var genes are bound by
• SIR2.
• SIR2 binding is lost when gene is activated.
• SIR2 gene disruption leads to activation of a
• subset of var genes.

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Layer 3 Subnuclear architecture
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Active Var Gene May Re-Locate to Region of
Euchromatin
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Clustering of var Genes May Promote Gene
Recombination
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Pathogenic Neisseriae
Gram-negative bacteria Two pathogens of
importance to human health N. Gonorrhoeae sexual
ly transmitted causes cervical and urethral
infections Uses pili to attach to epithelial
cells, invades, replicates in basement
membrane N. Meningitidis transmitted by saliva
or respiratory secretions cause of meningitis
Uses pili to attach to host cells
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Phase variation in Neisseria opa genes
Small set of outer membrane protein (Opacity
protein), up to 7 genes (possibly adhesive
proteins) All copies are transcribed, control is
at translation Signal sequences have variable
of coding repeats (CTCTT) Phase variation is
RecA independent, thought due to strand slippage
changes Very high frequency (gt10-2 per division)
ss
Opa
7 x CTCTT Stop 8 x CTCTT Stop 9 X CTCTT in
frame
CTCTTCTCTTCTCTTCTCTTCTCTTCTCTTCTCTTCTCTTCCGCA
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Antigenic Variation and Phase Variation of
Neisseria pili
Pathogen lifestyle extracellular and within
neutrophils Variant antigen type IV pilin
protein Protein location Expressed at surface
of bacteria Protein function Adhesion
ligand Gene copies one expressed gene (pilE),
several silent pseudogenes (pilS) Switch
rate as high as 4 X 10-3 per cell per
generation Role of recombination Antigenic
variation is RecA dependent
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Mechanisms of Pili Variation
Antigenic variation Occurs when silent pilin
cassettes (pilS) recombine with the sole
expression site (pilE) a) intergenomic
take up DNA released by lysis from
neighboring Neisseria
cells b) intragenomic recA dependent
recombination with silent copies Phase
variation the reversible inter-conversion
between piliated (P) and nonpiliated
states (P-). pilC is turned on or off.
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Organization of Pilin loci
Meyer et al. Clin Micro Reviews 1989. S139-145