dna repair and lupus - nih videocast · • germline variants in dna repair genes are linked to and...
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DNA Repair and LupusJoann B. Sweasy, Ph.D.
The Ensign Professor of Therapeutic Radiology Yale University School of Medicine
https://doi.org/10.1016/j.freeradbiomed.2016.10.507
Base Excision Repair
TDG),
DNA Damage and BER
• Bases damaged by RONs (respiration and inflammation)
• Uracil, deamination products• Methylated bases (endogenous processes,
environment, and chemotherapy)• Active demethylation• Somatic hypermutation• 20,000-50,000 lesions/cell/day
Pol β Plays a Critical Role in Base Excision Repair
C
G
ROS
=OG
Y265C Pol β is a Cancer-Associated Fidelity Mutant
• Slow polymerase. Extremely low catalytic efficiency.
• Has similar affinity as WT Pol β for single nucleotide gapped DNA.
• Mutator activity.• Induces anchorage independent
growth.
Screen for error-prone mutants
POLB somatic mutations ingastric carcinoma
Washington et al. PNAS 1997; Opresko et al, Biochem. ,1998; Clairmont et. al., PNAS, 1999
Y265C
C
GG
Pol β-Y265C Fills Gaps Slowly and in an Error-Prone Manner
C
A
E12 E13 E14*
Y265C
Is Y265C Pol β Associated With The Development of Cancer?
Senejani et al. PNAS 2012
Mice Deleted of POL B
• Perinatal lethality.• Massive apoptosis of postmitotic neurons.• Perinatal lethality not rescued by crossing into
TP53∆.• Embryonic lethality in a PRDCK∆ background;
high levels of neuronal apoptosis.
3 WEEKS
The POLBY265C/C Mice are Alive and Small
Senejani et al. PNAS 2012
Organs from C/C Embryos Exhibit High Levels of TUNEL Positive Cells
Cleaved Caspase-3
Cells from Cortex and Other Organs of C/C Embryos Undergo Apoptosis
Senejani et al. PNAS 2012
Senejani et al. PNAS 2012
Y265C/C MEFs Grow Slowly
OH5’3’
Pol βOH
5’3’dRP
MMS methylates G and A
Aag/Mpg
LigIIIα 5’
3’
Pol β
BER is Compromised in the C/C MEFs
dRP
Ape1
Senejani et al. PNAS 2012
OH5’3’
Pol βOH
5’3’dRP
MMS methylates G and A
Aag/Mpg
LigIIIα 5’
3’
Pol β
BER is Compromised in Bone Marrow
dRP
APE1
Senejani, unpublished
CBER
Intermediates
Low Levels of Gap Filling by Pol β-Y265C Lead to Accumulation of BER Intermediates
Double-strandBreaks?
Glycosylase/Lyase
*
Pol ß Y265C
Accumulation of BER Intermediates In Pol B-Y265C MEFs?
Increased Levels of SSBs are Observed in the C/C MEFs
WT WTC/C C/C
Senejani et al. PNAS 2012
Induced Levels of γH2AX Foci in the C/C MEFs Treated with MMS Suggest Increased DSBs
Senejani et al. PNAS 2012
Untreated C/C MEFs Have Increased Levels of DSBs
Senejani et al. PNAS 2012
Perc
ent W
ith γH
2AX
Foci
Glycosylase/Lyase
*
Pol ß Y265C
Accumulation of BER Intermediates In Pol B-Y265C MEFs
Apoptosis
Chromosomal Aberrations?
The C/C MEFs Display Increased Levels of Chromosomal Aberrations
Types of Aberrations
Aber
ratio
ns p
er C
ell
Types of AberrationsSenejani et al. PNAS 2012
Glycosylase/Lyase
*
Pol ß Y265C
Accumulation of BER Intermediates In Pol B-Y265C MEFs
Chromosomal Aberrations
Cancer?
Senejani et al. Cell Reports, 2014
The POLB Y265C/C Mice Develop Increased Levels of Dermatitis
The POLB Y265C/C Mice Develop Lupus-Like Dermatitis
Senejani et al. Cell Reports, 2014
The POLB Y265c/c and Y265c/+ Mice have Higher Levels of ANA than WT Mice
Senejani et al. Cell Reports, 2014
The POLBY265C/C Mice Have Higher Levels of Antinuclear Antibody
Senejani et al. Cell Reports, 2014
The POLBY265C/C Mice Exhibit Severe Glomerulonephritis
Senejani et al. Cell Reports, 2014
Immune Complexes in Kidneys of C/C Mice
H and E IgG
Senejani et al. Cell Reports, 2014
+/+
C/C
Clinical Manifestions of Systemic Lupus Erythematosus
• Dermatitis• High Antinuclear Antibodies• Renal Disease• Cervical Lymph Node Hyperplasia (mice)
The POLBY265C/C mice have lupus-like disease.
Senejani et al. Cell Reports, 2014
How is Inefficient and/or Error-Prone Gap Filling During BER Related to Lupus Development?
C
A
ImmunityDNA Repair VDJCSRSHM
Links Between DNA Repair and Immunity
V D J
A C
GT
ArtemisNicksHairpins
ATGC
GTAG
RAG Cleaves DNA
VDJ Recombination
ACGTAGTGCATC
GT
GA
The CDR3 Junctions are Short in the Heavy Chain in the POLB Y265C/C Mice
V D J
Intervening gene segments
V D J
WT: 27-60 bpC/C: 24-52 bp
Senejani et al. Cell Reports, 2014
ATGC
GTAG
ATGC
GTAGC
Pol β
V D
CTA ATGC
GTAG
ATGC
GTAG
A T
Pol β Y265C
Nuclease
The Role of Pol β is to Fill Gaps
Pol β functionsin end-joining
Ray et al. NAR, 2018
CG
UG
G
G
ACG
G
TT
AT
A
AID
MSH2/6EXO1/POLη
Mutationsat A:T
UDG APE1
Pol βTransversions/TLS Polymerase
Transitions/Replication
Somatic Hypermutation isCo-opted BER
Increased Frequency of Somatic Hypermutation in the POLB-Y265C Mice
p <1 x 10-11
Senejani et al. Cell Reports, 2014
CG
UG
G
G
A
G
TT
AT
A
AID
MSH2/6EXO1
Mutationsat A:T
UDG APE1
Transversions/TLS Polymerase
Transitions/Replication
X
Gaps May be Filled by a TLS Polymerase in POL B Y265C/C Mice
Y265C
WT
The Numbers of Germinal Centers are Increased in C/C Mice
Senejani et al. Cell Reports, 2014
Abnormal Gap Filling Leads to an Aberrant Antibody Repertoire
V D J
WT: 27-60 bpY265C: 24-52 bp
G
X
SHM
Pol ß Y265C
Cell Death
Slow Gap Filling Leads to Cell Death and Antigenic Debris in Replicating
CellsDSB
Cellular Debris
Y265C
WT
Senejani et al. Cell Reports, 2014
Cells are Dying in Germinal Centers of POLB-Y265C Mice
Summary• Pol β Y265C polymerase has low catalytic efficiency
and is error prone• Cells from POLB-Y265C mice exhibit genomic
instability• POLB-Y265C embryos exhibit massive apoptosis• POLB-Y265C mice are BER deficient• POLB-Y265C mice develop lupus
• Aberrant immune repertoire
Summary• Germline variants in DNA repair genes are linked
to and perhaps cause lupus development• DNA repair processes function in the
development of the immune repertoire• Defective DNA repair can result in mutations or
cell death, both of which can lead to the development of autoimmunity.
AcknowledgmentsSweasy LabAli SenejaniMatt BurakRithy MeasSesha Lakshmi PaluriSteven MaherTania Rahim
Lindsey CriswellJoanne NitithamKim Taylor
CollaboratorsMichael KashgarianCarolyn ZeissJennifer McNiffAlfred BothwellJoe CraftStephen Lloyd
R01 ES019179; R21AI124055
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