dna repair and lupus - nih videocast · • germline variants in dna repair genes are linked to and...

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


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



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


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Senejani, unpublished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


Induced Levels of γH2AX Foci in the C/C MEFs Treated with MMS Suggest Increased DSBs


Untreated C/C MEFs Have Increased Levels of DSBs


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


The POLB Y265c/c and Y265c/+ Mice have Higher Levels of ANA than WT Mice


The POLBY265C/C Mice Have Higher Levels of Antinuclear Antibody


The POLBY265C/C Mice Exhibit Severe Glomerulonephritis


Immune Complexes in Kidneys of C/C Mice

H and E IgG


+/+

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.


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


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


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


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


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

dna repair and lupus - nih videocast · • germline variants in dna repair genes are linked to and...

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