Balancinggyp Somatic Hypermutation and DNA Repair in ... · Balancinggyp Somatic Hypermutation and...
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Balancing Somatic Hypermutation and g ypDNA Repair in Immunoglobulin Genes
Patricia GearhartLaboratory of Molecular Gerontologyy gyNational Institute on Aging, NIH
Baltimore
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ANTIBODY (B cells)Light chain
ANTIBODY (B cells)
Heavy chain
Variable regions
Constant regions
Bind pathogen; Eliminate pathogen;Bind pathogen;hypermutation
Eliminate pathogen;switch recombination
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Activation-Induced Deaminase (AID)
deaminates cytosine to uracildeaminates cytosine to uracil
T C G A A G C T AID
T C G A A G U T
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U:G is mutagenic: strand breaks & mutations
A G U A G C TT C G T C G A
5’ 3’AID
BER MMR
A G A G C TT C G T C G A
5’ 3’UNG MSH2-MSH6
A G U A G C TT C G T C G A
5’ 3’2
AP endonuclease
A G A G C TT C G T C G A
5’ 3’
Exonuclease 16
A G U A G C T5’ 3’2
DNA polymerase Rev1
T C G T C G A
A G N A G C T5’ 3’
DNA polymerase η
T C G T C G A6
A G C N G C T5’ 3’
Poleta
A G N A G C TT C N T C G A
G:C mutationsSt d b k
A G C N G C TT C G T C G A
A:T mutationsSt d b k
NeubergerStavnezerJacobs
Strand breaksGearhartScharffReynaud
Strand breaks
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I The Master CatalystI. The Master Catalyst
G II. Going Rogue
III. The Targeting Enigma
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I. AID - the Master Catalyst
Protein loop that recognizes the AID hot spot: WRC (W = A/T; R = A/G)
Rahul Kohli, JHU
JBC 284:22898-04 (2009)
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DNA cytosine deaminases
Antibody diversity: AID WRCAntibody diversity: AIDHIV retroviruses: APOBEC 3F
APOBEC 3G
WRCTTCCCC
How do they recognize different motifs?
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Structural alignment suggests a “h t t iti l ”“hotspot recognition loop”
H th i G fti l f th APOBEC3 Hypothesis: Grafting loops from the APOBEC3 enzymesinto AID will lead to predictable changes in specificity
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Purify variant deaminases from bacterial cultures. Incubate with oligonucleotides containing XXC (X = A, mC, G, or T).
X-2 X-1C5’ 60 nt 3’
Deaminase
XXU
Uracil DNA glycosylase g y y
XX
Abasic endonuclease ^
24 nt 36 nt
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AID/APOBEC loop graft variants change AID/APOBEC loop graft variants change sequence motifs as predicted
AID WRC
AID 3FL TTCAID-3FL TTC
AID-3GL CCC
Are motifs altered in E. coli?
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rpoBrifampin deathrpoB death
rifampin Resistant colonies;sequence rpoB gene
rpoBAID
deaminaserpoBX
WRC
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9 amino acid loop in AID is responsible for recognizing hot spot motif WRC.
Similar loops in APOBEC3F/G can be transferredto change specificity of AID. to change specificity of AID.
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II Going Rogue Uracils in DNAII. Going Rogue – Uracils in DNA
AID proposed to deaminate RNA, whereas genetic and biochemical D propos to am nat N , wh r as g n t c an och m ca data support DNA deamination
…but, no evidence for uracils in immunoglobulin loci.
Uracils in variable region DNA from B cells
R b MaulRob Maul
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Digest DNA (Ung-/- Peyer’s patch B cells) with l l l ( ) d d l ( E)uracil glycosylase (UDG) and abasic endonuclease (APE)
ConstantVariable U UU U Constant
UDG
APE
PCR amplify
XXHypothesis: more uracils = less amplification of V gene
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Location of primers for nested PCR
VDJ 8 kbCμ
VDJ Cμ
k5 kbVκ Cκ
Vκ Cκ
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PCR assay
pg DNA 300 300 100 30
Ung-/- Ung-/-Aid-/-
y
300 300 100 30UDG, APE - + + + - + + +
VDJ
Cμ
Vκ
More UDG-sensitive sites in V genes than C genes;
Cκ
More UDG sensitive sites in V genes than genes;AID dependent.
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III The Targeting EnigmaIII. The Targeting Enigma
Setting up the Sμ locus for AID
Rob Maul Deepa Rajagopal
JEM 206:1237-44 (2009)
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Peaks of hypermutation after V and Iμ promoters
10-2
4
10-6
10-4
10-8
Does location of RNA pol II correlate with this pattern?
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H w d es DNA structure in S affect transcripti n?
5 kb0 2 4 1 3
How does DNA structure in Sμ affect transcription?
E H H H
S7 S8 S9S1 S2 S3 S4 S5
Eμ Sμ RR Cμ1
S7 S8 S9S1 S2 S3 S4 S5
GGGGAGCTGGGGAGCTGGGClusters of G and AGCT hotspots for AIDp
R-Loop RegionRNA pol II
DNA-RNA hybrid
Lieber
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Map the position of RNA pol II by nuclear run-on
Stimulate Ung-/- splenic B cells with LPS + IL4. After 2 days, isolate nuclei.
Addition of
g p y ,
ll
Gentle Lysis[32P]UTP, ATP,
CTP, GTP
cells nuclei transcription
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Position of RNA pol II molecules
S7 S8 S9S1 S2 S3 S4 S5
Eμ Sμ RR Cμ1
tens
ity Ung-/-
Day 06
8
10S1S2S3
β-actCD3δ
Rel
ativ
e in
t y
0
2
4S3S4S5S7 S8
S9
S1 S2 S3 S4 S5 S7 S8 S9 γ-act β-act CD3δ
Ung-/-
Day 2
ativ
e in
tens
ity
2
4
6
8
10S1S2S3S4
β-actCD3δ
γ-act
Rel
a
0
2
S1 S2 S3 S4 S5 S7 S8 S9 γ-act β-actCD3δ
S5S7 S8
S9
P l f l h d f h Pileup of polymerases on either side of the repetitive region.Does not depend on cell activation.
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Aid-/-Ung-/-y10
S1 β-actAid UngDay 2
Rel
ativ
e in
tens
ity
2
4
6
8S2
S3
S4S5
CD3δ
S8
S9
R 0 S7 S8S1 S2 S3 S4 S5 S7 S8 S9 γ-act β-act CD3δ
C
S1 β-actSμ dely 8
10
S1 S2 ∆S Cμ
Eμ Cμ1
S2
∆S
Cμ
CD3δ
t
Sμ delDay 2
Rel
ativ
e in
tens
it
0
2
4
6
8
Pileup does not depend on AID. Depends on DNA sequence of Sμ.
Cμ γ-act0S1 S2 ∆S Cμ γ-act β-act CD3δ
p p p q
RNA pol II location confirmed by ChIP
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RNA pols pause in R-loops
GGGGAGCT GGGGAGCT GGGGAGCT
p p p
CCCCTCGA CCCCTCGA CCCCTCGA
CμSμ coreiEμR-loop
RNAP RNAP RNAP
Fast TranscriptionSlow Transcription- High pol densityFast Transcription
- Low pol densityHigh pol density
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How does DNA structure affect somatic hypermutation?
Sequenced JH4 intron DNA from immunized B cells from Ung-/- miceSequenced JH4 intron DNA from immunized B cells from Ung mice
8
10
y (x
10-3
)
4
6
on F
requ
ency
0
2
Mut
ati
not sequenced
Sμ RREμ
Mutations increase before the repetitive region Mutations increase before the repetitive region and decrease after
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DNA structure pauses RNA pol II and affects mutationDNA structure pauses RNA pol II and affects mutation
CμRNA pol II
CμAID
AID may associate with RNA pol II. R-loop regions pause RNA AID may associate with RNA pol II. R loop regions pause RNA polymerases, increasing AID binding to ssDNA and mutations. After core, AID falls off or is diluted out, and mutation stops.
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What targets AID to V region?g g
Cμ CγS S
DSB DSB
V
NHEJIgM → IgGgM g
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Rob MaulHuseyin SaribasakRhonda McClure
Rahul KohliJim StiversDavid WilsonRhonda McClure
Zheng CaoWilliam Yang
David WilsonRanjan SenRoger Woodgate