This document discusses mechanisms of genetic recombination and antigenic variation. It begins by explaining the benefits of recombination, including separating favorable and unfavorable mutations and generating new gene combinations. It then describes several types of homologous recombination, including double-strand break repair and gene conversion. Specific proteins involved in initiating recombination, such as RecBCD and RecA, are also discussed. The document focuses on examples of antigenic variation in pathogenic protozoa, including Plasmodium falciparum and African trypanosomes. It describes how these organisms use large gene families and recombination-mediated switching of variant surface antigens to evade the immune system through clonal antigenic variation.

1. Recombination, Phase Variation and
Antigenic Variation

2. Why Recombination?
Two Broad Categories
Homologous (or general)
Site-specific (e.g. phage genomes into bacterial chromosomes)
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

3. 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)

4. General Recombination often involves a
Holliday Junction
Proposed by Dr. Holliday (Holliday R. 1964. A mechanisms for gene conversion in fungi.
Genet. Res. 5:282-304)
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

5. Holliday structures (part I)
Sister chromotids
Sister chromotids
e.g. 4 strand
during meiosis

6. Holliday structures (part II)

7. Recombination is initiated by
double-strand breaks in DNA

8. a a b b
A
B
A B
a+ b
a +B
Mechanisms of Gene
Conversion/Recombination
1) Double-strand break (DSB) repair
2) Synthesis-dependent strand annealing (SDSA)
Chen J et al. Nature Reviews Genetics. 2007. 8:762-775

9. 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)

10. 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

11. 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
RecBCD

12. Initiation of recombination by the
RecBCD enzyme

13. 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
RecA enables single stranded DNA
to invade DNA helix
Eukaryotes have multiple homologs of bacterial RecA
(Rad51 is best studied)

14. Chi site Χ
Recombination hotspot
Modifies RecBCD enzymatic activity
5’ GCTGGTGG 3’
1009 chi (Χ) sites in E. coli genome
Χ homologs in other bacteria

15. Kowalczykowski TIBS. 2000. 25: 156-65

16. Targeted gene disruption by
homologous recombination
Lodish et al. Molecular Cell Biology

17. Gene Conversion
A special type of homologous recombination
Non-reciprocal transfer of genetic material from a ‘donor’
sequence to a highly homologous ‘acceptor’ sequence
Initiated by double strand DNA (dsDNA) breaks
5’ > 3’ exonucleases
3’ ssDNA tail strand invasion (RAD51 and others)
Outcome: portion of ‘donor’ sequence copied to ‘acceptor’
and original ‘donor’ copy unchanged
donor acceptor
gene
conversion

18. 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.

19. Clonal Antigenic Variation
in Pathogenic Protozoa

20. Variable Surface Antigens of Pathogenic Protozoa
Organism
Causative
Agent
Variable
Antigen
Structural Features Functions
Plasmodium
falciparum
Malaria
PfEMP1 (var
gene product)
200-350 kDA
transmembrane
protein at the surface
of IRBC
Infected erythrocyte surface,
adherence to host surface molecules
African
Trypanosom
es
African
Sleeping
sickness
VSG
~60 kDa protein
anchored by GPI-
linkage at the parasite
surface
Densely packed, variable surface
coat, immune evasion
Two Pathogens: Two Approaches

21. 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
Recombination generates diversity in gene families
Means for survival and transmission
Common Themes of Clonally Variant Antigens
Kyes S. et al. Annu Rev. Microbiol. 2001. 55:673-707

22. 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
Variations on a Theme

23. Clonal Antigenic Variation in Trypanosoma brucei
Ab surface labeling of VSGs
from a mixed population

24. Trypanosome antigen switching
At each wave, different VSGs are expressed
Switch rates - 10-2 to 10-6 per cell in blood
>100 VSGs expressed sequentially in one rabbit
Switch not induced by the immune system
Semi-programmed -- early VSGs are always early

25. 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

26. VSG Proteins Have Diverse Sequence
but Related Structure
Blum M et al. Nature. 1993. 362: 603-9
• 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.

27. 7 6 5 4 8 3 2 1
VSG
70-bp
repeat
Telomeric
repeats
ESAGs
a-amanitin resistant
Pol I promoter
VSG Expression Occurs From Unique
Telomeric Expression Sites
• VSG are expressed in long polycistronic messages (>40kb)
• 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
1. Bloodstream (above)
2. metacyclic

28. VSG
ESAGs
VSG
ESAGs
One VSG Expression Site is Active at a Time
Active Site
(one)
Inactive
‘silent’ site
(many)
Full-length transcript, high level
Partial transcripts, low level
70-bp
repeat
70-bp
repeat
Unable to “force” two expression sites to be simultaneously active
Chaves et al 1999. EMBO J 18:4846-55

29. Active VSG locus
tagged with
Lac operator and
Visualized with
Tagged lac
Repressor LacI-GFP
Nucleolus and
“Expression
Site Body (ESB)”
Labeled by
PolI antibody
ESB ESB
Nucleolus
Active VSG is Located in Subnuclear Compartment
“Expression Site Body”
Navarro M & Gull K. 2001. Nature 414:759-763.

30. Three Distinct Mechanisms of VSG switching
Duplicative Gene
Conversion
A
221
A
A
in situ
switch
221
221
C
C
Telomere
exchange
B
221
221
B

31. Location VSG
Size VSG pool
Silent subtelomeric
VSG arrays
Telomeric VSGs VSGs in bloodstream
expression sites
1250-1400 150-250 20
Megachromosomes and
intermediate chromosomes
Minichromosomes
~50-100kb
VSG Genome Organization
Taylor & Rudenko. Trends Genet 2006. 22:614-20

32. 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
vsg Gene Diversification

33. Silent
VSG array
Active VSG
expression site
3’conserved
region
70-bp repeat
A B
B
C
70-bp repeat arrays
VSG Switching by Gene Conversion Frequently Relies on Homology
Upstream and Within the 3’ Conserved Region of Genes
Switch

34. L Miller et al, Nature 2002
Clonal Antigenic Variation in Plasmodium falciparum

35. Plasmodium falciparum: Antigenic
Variation and Cytoadhesion are Linked
PfEMP1 switch,
binding/antigenicity
changes
Miller 2002

36. ~60 genes per parasite haplotype (few pseudogenes)
One gene on, the others off
Similar A,B,C gene organization between parasite isolates
A’s are not under strong CD36 selection, others are
The A,B,Cs of var
Organization

37. 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

38. Multiple Layers of Gene Control
Layer 1: Gene structure and putative regulatory
elements

39. Var Genes are Expressed in situ
DBL CIDR DBL DBL ATS
TM
Sterile Transcripts
Exon 1
hypervariable
Exon 2
Conserved
• 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?

40. var Intron Promoter May Cooperate in Gene Silencing
luciferase
var upstream region
luciferase
var upstream region
luciferase
var upstream region
intron promoter
disabled intron promoter
Default var
promoter state
Active
Active
Silent
promoter pairing
Deitsch et al. Nature. 2001. 412:875-6

41. 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

42. Layer 2: Chromatin modifications

43. Silent and Active Chromatin Marks
Note: similar “marks” are found at many active and silent genes

44. 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.

45. Layer 3: Subnuclear architecture

46. Active Var Gene May Re-Locate to
Region of Euchromatin

47. Clustering of var Genes May Promote Gene
Recombination

48. Pathogenic Neisseriae
Gram-negative bacteria
Two pathogens of importance to human health
N. Gonorrhoeae
sexually 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

49. 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 (>10-2 per division)
Opa ss
…CTCTTCTCTTCTCTTCTCTTCTCTTCTCTTCTCTTCTCTTCCGCA…
7 x CTCTT = Stop
8 x CTCTT = Stop
9 X CTCTT = in frame
Phase variation in Neisseria opa genes

50. 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

51. 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.

52. Organization of Pilin loci
Meyer et al. Clin Micro Reviews 1989. S139-145