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
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
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
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
I The Master CatalystI. The Master Catalyst
G II. Going Rogue
III. The Targeting Enigma
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)
DNA cytosine deaminases
Antibody diversity: AID WRCAntibody diversity: AIDHIV retroviruses: APOBEC 3F
APOBEC 3G
WRCTTCCCC
How do they recognize different motifs?
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
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
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?
rpoBrifampin deathrpoB death
rifampin Resistant colonies;sequence rpoB gene
rpoBAID
deaminaserpoBX
WRC
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.
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
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
Location of primers for nested PCR
VDJ 8 kbCμ
VDJ Cμ
k5 kbVκ Cκ
Vκ Cκ
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.
III The Targeting EnigmaIII. The Targeting Enigma
Setting up the Sμ locus for AID
Rob Maul Deepa Rajagopal
JEM 206:1237-44 (2009)
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?
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
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
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.
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
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
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
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.
What targets AID to V region?g g
Cμ CγS S
DSB DSB
V
NHEJIgM → IgGgM g
Rob MaulHuseyin SaribasakRhonda McClure
Rahul KohliJim StiversDavid WilsonRhonda McClure
Zheng CaoWilliam Yang
David WilsonRanjan SenRoger Woodgate