molecular mechanisms of mismatch repair · purified human proteins (muts , muts , mutl , exo1, rpa,...
TRANSCRIPT
11/21/07
Molecular Mechanisms of Mismatch Repair
Guo-Min Li
Departments of Toxicology and BiochemistryMarkey Cancer Center
University of Kentucky College of Medicine
11/21/07
Mismatch Repair Deficiency Causes Cancer
Mismatch repair (MMR) deficiency(Genetic defects or epigenetic
modification of MMR genes)
Genomic instability
(e.g., Microsatellite instability)
Cancer
(e.g., HNPCC or Lynch Syndrome)
11/21/07
MMR Genes and Colorectal Cancer
MSH2
HNPCCa Sporadic
a Hereditary non-polyposis colorectal cancer.b Hypermethylation of the MLH1 promoter.
~40%
~50%
<5%
70%
30%
MLH1b
0%
Population incidence ~1 in 500 1 in 20
Microsatellite instability >90% 13%
MMR gene mutations 70% ~65% of CRC with MSI
MSH3MSH6
PMS1
PMS2MLH3
~0.5%
11/21/07
Genome-Maintenance Functions of MMR
I. Correct biosynthetic errors
II. Suppress homeologous recombination
III. Mediate DNA damage response
11/21/07
DNA Mismatch Repair Reaction
Repair synthesis
Mismatch removal
Mismatch recognitionMutS homologs
MutL homologs
Nuclease
Helicase
Replicative DNA polymerases
Parental strand
Daughter strand
11/21/07
Mismatch Repair Components
E. coli
MutS
MutL
MutH
UvrD (helicase II)
RecJ, ExoI, ExoVII, ExoX
SSB
DNA pol III holoenzyme
DNA ligase
Human
MutS (MSH2-MSH6)
MutS (MSH2-MSH3)
MutL (MLH1-PMS2)
?
?
EXO1, ?
RPAPol , PCNA, RFC
DNA ligase I
HMGB1
11/21/07
Outlines
• Identification and characterization of mismatch
repair components
– RPA
– HMGB1
– PCNA
• Reconstitution of the mismatch repair reaction
• Identification and characterization of mismatch
repair components
– RPA
– HMGB1
– PCNA
• Reconstitution of the mismatch repair reaction
11/21/07
Fractionation
HeLa nuclear extract
0-35 36-65
(HN ) SO4 2 4
FI FII
ssDNA cellulose
SS1 SS2
11/21/07
In vitro Mismatch Repair Assay
TTCGAGAGCTC 5’AAGCTTTCGAG 3’
XhoI
HindIII
11/21/07
Fractionation and Reconstitution
HeLa nuclear extract
0-35 36-65
(HN ) SO4 2 4
FI FII
ssDNA cellulose
SS1 SS2
11/21/07
Purification of the SSI/FII-Complementing Activity
212
97
158116
67
27
20
14
73.5
37
5743
Fraction # 23 26 29 32 35 38 41 44 47 50 53 56 59 62 65 68 FT
SSI + FII
SS2 on Mono Q FPLC columnSDS PAGE of fraction 65
11/21/07
Recombinant RPA Substitutes for SS2
Repair
products
HeLa
SS1
SS2
FII
RPA
+
-
-
-
-
-
+
-
-
-
-
+
+
+
-
-
+
-
+
+
-
+
-
-
+
Ramilo et al., Mol. and Cell. Biol. 22, 2037-2046, 2002
11/21/07
RPA Protects Nascent ssDNA?
BseRI
11/21/07
RPA Protects Nascent ssDNA
HeL
a
SS1
RPA (μg)
0 0.5 1 2
SSB (μg)
Bla
nk
2 4 8HeL
a/A
PD
Bsp106
Ramilo et al., Mol. and Cell. Biol. 22, 2037-2046, 2002
11/21/07
RPA Is Phosphorylated during MMR
Nuclear extract
Incubation
Protein-DNA
complex
Proteins eluted
SDS PAGE
DNA substrate
Elution
Western blotting
0 0.5 1 2 4 6 10 20
Reaction time (min)
MSH2MLH1pol
PCNARPA1
RPA2
G-T
A-T
MSH2MLH1pol
PCNA
RPA1
RPA2
ExcisionResynthesis
Unphosphorylated RPA for excision?
Phosphorylated RPA for resynthesis?
11/21/07
Unphosphorylated RPA Facilitates Excision
RP
A
RP
A2A
RP
A2D
60 Excision (%)45 27
Product
11/21/07
Phosphorylated RPA Promotes Resynthesis
WT
2A
2D
No RPA
11/21/07
Phosphorylation Reduces RPA DNA Binding
11/21/07
Phosphorylation Regulates RPA Functions?
5’
3’
3’
5’
5’
3’
3’
5’
5’
3’
3’
5’
5’
3’
3’
5’
5’
3’
3’
5’
Initiation
Excision
Resynthesis
Phosphorylated RPA
Unphosphorylated RPA
EXO1
Pol
11/21/07
Role of RPA in MMR
1. Involved in all steps of MMR
2. Protect the nascent ssDNA from degradation
3. Phosphorylation may regulate RPA functions in
MMR
11/21/07
Role of HMGB1 and PCNA in MMR
Yuan et al., J. Biol. Chem. 279, 20935-20940, 2004Guo et al., J. Biol. Chem. 279, 16912-7, 2004.Gu et al., Nucleic Acids Res. 26, 1173-1178, 1998
HMGB1:Involved in the excision step in MMR
PCNA:1. Co-IP with MSH2 and MLH1
2. Involved in both the steps of excision and resynthesis in
MMR
3. Differentially required for 3’ and 5’ directed MMR
11/21/07
Outlines
• Identification and characterization of mismatch
repair components
– RPA
– HMGB1
– PCNA
• Reconstitution of the mismatch repair reaction
11/21/07
Unsolved Fundamental Problems
Resynthesis
Excision
Mismatch recognition
How does mismatch recognition
by MutS homologs trigger the
downstream repair events?
How is mismatch-provoked
excision terminated upon
mismatch removal?
11/21/07
Initiation Models
Translocation
Sliding clamp
MutL, ATP MutH
Moving
or Cis
Stationary
or Trans
# of Protein complex
1
More than 1
1
11/21/07
Purification of MMR Proteins
11/21/07
Reconstitution of MMR Reaction
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Pol
PCNA
RFC
HMGB1
RPAMutL
MutS
EXO1
+
+
+
+
+
+
3.3 kb3.1 kb
11/21/07
Stoichiometry of MMR Reaction
• Protein concentrations were based on the amount of individualproteins in 50 μg of HeLa nuclear extracts:
– 400 fmol MutS
– 300 fmol MutL
– 800 fmol RPA
– 1.2 pmol HMGB1
– 5 fmol EXO1
• 24 fmol heteroduplexes
11/21/07
Multiple MutS /MutL Proteins Are Required forRepair of a Single Mismatch
11/21/07
Initiation Models
Translocation
Sliding clamp
MutL, ATP MutH
Moving
Stationary
?
11/21/07
Role of MutL in MMR
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Pol
PCNA
RFC
HMGB1
RPAMutL
MutS
EXO1
+
+
+
+
+
+
3.3 kb3.1 kb
11/21/07
Role of MutL in MMR
+ M
utL
- MutL
HindIII/BspDI
HindIII/BspDI
Resynthesis
Resynthesis
11/21/07
Extensive Excision without MutL
SspI
SspI
11/21/07
MutL Terminates Excision uponMismatch Removal
11/21/07
Model for Mismatch-Provoked Excision
11/21/07
Summary
• Reconstituted the 5’ nick-directed mismatch repair using 10purified human proteins (MutS , MutS , MutL , EXO1, RPA,
HMGB1, pol , PCNA, RFC, and DNA ligase I).
• MutL acts to terminate mismatch-provoked excision upon
mismatch removal.
• More than one MutS -MutL complex is required for the
repair of a single mismatch.
11/21/07
Reconstitution of Human MMR
Zhang et al., Cell, 122, 693-705, 2005
11/21/07
Acknowledgement
Yanbin Zhang
Fenghua Yuan
Cecilia Ramilo
Shuangli Guo
Zhanmin Fan
Steven Presnell
Keli Tian
Yin Gao
Guogen Mao
Caixia Hou
Lei Tian
Nelson Chan
Janice Rodriguez
Zhaolong Li
Leon Lu
Jianxin Wu
Scott McCulloch
Beibei Zhu
Brandee Brown
Hiroaki Murata
Liya Gu, University of Kentucky
Isaac Wong, University of Kentucky
Mike Liskay, Oregon Health & Sciences University
Marc Wold, University of Iowa
Joe Jiricny, University of Zurich, Switzerland
Bruce Stillman, Cold Spring Harbor Laboratory
Carol Prives, Columbia University
Ellen Fanning, Vanderbilt University
Lene Rasmussen, Roskilde University, Denmark
Alan Tomkinson, University of Maryland, Baltimore
John Turchi, Indiana University School of Medicine
NIH for funding