Biochemical Analysis of XRCC1

Interactions with BER enzymes

Claire Cato

April 23, 14

Department of Biological Sciences

Eichman Lab

DNA is subject to constant damage

Environmental toxins

Endogenous metabolites

Replication errors

Base alkylating drugs

DNA Damage

Replication Fork Arrest

Mutations

Direct reversal

Oxidative demethylation

Base excision

Nucleotide excision

Recombination

DNA Repair

Genetic Instability

Cell Death

Cancer

Methyl Methanesulfonate

(MMS)

Methylnitronitrosoguanidine

(MNNG)

Alkylation damage is important for the discovery of DNA repair

mechanisms

BER removes small alkylation and oxidation damage

XRCC1 is involved in DNA repair but has no catalytic function

Mutagen

Sensitivity

Screens

DNA glycosylase

DNA ligase DNA polymerase

AP endonuclease

BER enzymes

X-ray repair cross-

complementing protein 1

(XRCC1)

XRCC1 contains two BRCT protein-protein interaction domains

BRCT = “BRCA1 C-terminus”

BRCT found in >50 proteins

XRCC1 Binding Partners

BRCT2 binds to DNA ligase III (Caldecott 1995)

NTD interacts with DNA polymerase β (Kubota 1996)

BRCT1 interacts with AP endonuclease I (Vidal 2001)

NTD binds to BER intermediate DNA structures (Nazarkina 2007)

Recent work shows BRCT1 interacting with DNA glycosylases (Campalans 2005)

NTD BRCT1 BRCT2

1 183 315 407 538 633

Hinge1 Hinge2

Amylose

MBP XRCC1

MBP XRCC1

A

A

A

MBP XRCC1

Amylose MBP XRCC1

A

A

A

Amylose MBP XRCC1

Amylose MBP XRCC1

1. Reaction Mix 2. Add 3. Isolate MBP-fusion

protein

Affinity chromatography is used to isolate MBP-fusion proteins

and their binding partners

Amylose MBP XRCC1

A

Amylose MBP XRCC1

A

Amylose MBP XRCC1

A

XRCC1 “pulls down” human 3mA DNA glycosylase (AAG) activity

AAG

Campalans et al. DNA Repair 2005 Marsin et al. JBC 2003

No AAG excision

AAG excision

MBP-XRCC1 AAG XRCC1-His

- + + -

- - + +

- - - +

XRCC1 MBP A Amylose + +

Amylose XRCC1 MBP

A

XRCC1 is a proposed BER scaffolding protein

Proposed Roles of XRCC1

1. Recruit BER enzymes to sites

of damage

2. Shield cell from toxic DNA

intermediates

3. Stimulate BER enzyme activity

4. Hand-off mechanism

Vidal et al. EMBO Journal 2001

How does XRCC1 interact with AAG?

Project Plan

1. Purify MBP-fused XRCC1 domains

1. BRCT1

2. Hinge-BRCT1

2. Perform affinity chromatography to verify the proposed interaction

with human AAG (Δ79AAG)

3. Determine reaction rates of AAG in the presence and absence of

XRCC1 domains

4. Crystallize AAG in complex with MBP-BRCT1 or MBP-Hinge-BRCT1

NTD BRCT1 BRCT2

1 183 315 407 538 633

Hinge1 Hinge2

BRCT1 MBP

BRCT1 MBP

AAG has a weak in vitro interaction with BRCT1

MBP-BRCT1+AAG

MBP-BRCT1

AAG

MBP-BRCT1+AAG+DNA LpxA+AAG

LpxA

MBP-BRCT1

AAG AAG

Load Elute Beads Load Beads Load Elute Beads Elute

AAG excision of εA is monitored using a glycosylase assay

1,N6-ethenoadenine (εA)

DNA denaturing gel

DNA glycosylase NaOH εA

Glycosylase Assay

εA

Preliminary results suggest AAG activity is enhanced by BRCT1

t (min)

S

P

0 240 0 2 5 15 30 60 240 0 2 5 15 30 60 240 0 2 5 15 30 60 240

No Enz AAG AAG+BRCT1 AAG+Hinge-BRCT1

0

0.2

0.4

0.6

0.8

1

0 50 100 150 200 250

Glycosylase Assay+/- XRCC1

Trial 2 - 140418 No EnzymeAAGAAG+MBP-BRCT1AAG+MBP-HB1

y = 0.023129 - 1.3693e-05x R= 1

Time (min)

y = m3-m1*exp(-m2*m0)

ErrorValue

0.0120680.94463m1

0.00128520.03721m2

0.0104120.96288m3

NA0.00052421Chisq

NA0.9997R

y = m3-m1*exp(-m2*m0)

ErrorValue

0.0110720.7913m1

0.00165070.042487m2

0.00938160.96371m3

NA0.00046035Chisq

NA0.99963R

y = m3-m1*exp(-m2*m0)

ErrorValue

0.0125580.93923m1

0.00152730.041352m2

0.0106850.96236m3

NA0.00058716Chisq

NA0.99966R

0

0.2

0.4

0.6

0.8

1

0 50 100 150 200 250

Glycosylase Assay+/- XRCC1

Trial 2 - 140418 No EnzymeAAGAAG+MBP-BRCT1AAG+MBP-HB1

y = 0.023129 - 1.3693e-05x R= 1

Time (min)

y = m3-m1*exp(-m2*m0)

ErrorValue

0.0120680.94463m1

0.00128520.03721m2

0.0104120.96288m3

NA0.00052421Chisq

NA0.9997R

y = m3-m1*exp(-m2*m0)

ErrorValue

0.0110720.7913m1

0.00165070.042487m2

0.00938160.96371m3

NA0.00046035Chisq

NA0.99963R

y = m3-m1*exp(-m2*m0)

ErrorValue

0.0125580.93923m1

0.00152730.041352m2

0.0106850.96236m3

NA0.00058716Chisq

NA0.99966R

0

0.2

0.4

0.6

0.8

1

0 50 100 150 200 250

Glycosylase Assay+/- XRCC1

Trial 2 - 140418 No EnzymeAAGAAG+MBP-BRCT1AAG+MBP-HB1

y = 0.023129 - 1.3693e-05x R= 1

Time (min)

y = m3-m1*exp(-m2*m0)

ErrorValue

0.0120680.94463m1

0.00128520.03721m2

0.0104120.96288m3

NA0.00052421Chisq

NA0.9997R

y = m3-m1*exp(-m2*m0)

ErrorValue

0.0110720.7913m1

0.00165070.042487m2

0.00938160.96371m3

NA0.00046035Chisq

NA0.99963R

y = m3-m1*exp(-m2*m0)

ErrorValue

0.0125580.93923m1

0.00152730.041352m2

0.0106850.96236m3

NA0.00058716Chisq

NA0.99966R

0

0.2

0.4

0.6

0.8

1

0 50 100 150 200 250

Glycosylase Assay+/- XRCC1

Trial 2 - 140418 No EnzymeAAGAAG+MBP-BRCT1AAG+MBP-HB1

y = 0.023129 - 1.3693e-05x R= 1

Time (min)

y = m3-m1*exp(-m2*m0)

ErrorValue

0.0120680.94463m1

0.00128520.03721m2

0.0104120.96288m3

NA0.00052421Chisq

NA0.9997R

y = m3-m1*exp(-m2*m0)

ErrorValue

0.0110720.7913m1

0.00165070.042487m2

0.00938160.96371m3

NA0.00046035Chisq

NA0.99963R

y = m3-m1*exp(-m2*m0)

ErrorValue

0.0125580.93923m1

0.00152730.041352m2

0.0106850.96236m3

NA0.00058716Chisq

NA0.99966R

BRCT1 MBP

BRCT1 MBP

The data suggest a weak interaction between MBP-XRCC1

domains and AAG

Weak binding under in vitro conditions

• Use different constructs

• Full length XRCC1

• Full length AAG

• Tag other than MBP

Post-translational modifications

• Express proteins in a eukaryotic

system

• Use antibodies to detect specific

PTMs

Weak interaction between AAG and XRCC1 may be a

biologically relevant observation

• Transient interaction between AAG and XRCC1 suggests

a hand-off mechanism over a stable BER complex

• Or may be regulated by the environment of the cell or

state of the DNA

Acknowledgements

Dr. Brandt Eichman

Suraj Adhikary

Sonja Brooks

Wenyue Du

Briana Greer

Lyla Kotsch

Dr. Aaron Mason

Dr. Elwood Mullins

Kevin Pereira

Rongxin Shi

Diana Tafoya

Eichman Lab