Transcript Biochemical Defects Associated with Cancer

Biochemical Defects Associated with Cancer-Causing Pathogenic
Mutations in Human MLH1
Andrew Nguyen
Laboratory of Dr. Andrew Buermeyer
Department of Environmental and Molecular Toxicology
Oregon State University
Deaths Caused by Cancer – United States, 2005
Men
291,270 deaths
Women
273,560 deaths
Colon and rectum
10%
Approximately 56,000 deaths from
colorectal cancer in 2005 alone
©2005 American Cancer Society, National Cancer Institute
Why DNA repair is important
DNA can be damaged by mutagens (ex: UV light, Radiation, Chemicals)
DNA replication is error prone – base pair mismatches are sometimes introduced by DNA
Polymerase
Lack of correction can lead to mutations, a risk factor for cancer
Consequences of DNA Mismatch Repair Deficiency
Accumulation of mutations in DNA can cause cancer.
Loss of mismatch repair (MMR) is linked to significantly increased cancer risk in
a condition known as Lynch Syndrome. This disorder is characterized by a predisposition
to early onset of colorectal and other internal cancers.
Knowing who is at risk for cancer because of MMR deficiency can influence treatment
options for existing patients or surveillance strategies for higher risk people.
DNA Mismatch Repair (MMR)
Evolutionarily conserved process
Mechanism involves several protein dimers
necessary for successful repair
MutLα is composed of two separate proteins,
MLH1 and PMS2
MMR acts to maintain genomic integrity by correcting DNA replication errors
Mechanism of Mismatch Repair
In-Vitro strand choice is determined
by nick in non-template strand
G
Recognition
MutS
T
MutL
Exonuclease
Excision
T
A
DNA polymerase
Resynthesis
T
Project Goals
Goals: Establish experimental conditions for an in-vitro repair assay
Compare four MLH1 mutants identified in human cancers vs. wild type MLH1
to see how/if the mutations affect MMR
These mutants are: K751R, R755W, L582V, L607H
[original amino acid] [location of mutation] [new amino acid]
Approach: In-vitro repair assay with MP1 extract (MutLα deficient) plus recombinant
MutLα
Questions: What level of DNA repair activity will the mutants exhibit compared to the wild type?
Amount of substrate repaired or kinetics of repair, for example
Experimental Approach
A plasmid containing a single known mismatch will be the substrate for the repair assays
Recognition and correction of mismatch will restore the Xho1 restriction site
on the plasmid
MMR activity assessed by percentage of plasmid substrate that can be restricted
Resynthesis
Excision
Ban1 restriction site
Ban1 restriction site
Restriction
Ban1 restriction site
0
50 100
% repair
Creation of Mismatch Containing Plasmid Substrate
Start with the pRO1 plasmid
pRO1
Nick plasmid
Generate gap by adding excess of
complementary oligonucleotide
Anneal a DNA fragment of known
sequence that will create a mismatch
Mismatch substrate
Experimental Approach – MP1 titration
Question: How much non-MutL containing cell extract is saturating?
MP1 cellular extract contains all MMR factors that are required for repair except MutLα
A titration was done to identify conditions under which these factors aren’t limiting for repair
Repair efficiency CT loop
80.00
60.00
% repair
0
70.00
No
MutL
50.00
40.00
pk height
30.00
20.00
10.00
Increasing amounts of MP1 added
0.00
0
50
100
150
200
250
300
350
ug mp1
Repair is MutL, MP1 dependent
Repair saturated around 225-250 µg
Experimental Approach – MLH1 WT Kinetics
Question: How does repair progress with time?
MLH1 WT Kinetics
0
5
Minutes
10 15 20 25 30 35 40 45 50 55 60
80
70
% repair
60
50
40
Time points
30
20
10
Identical repair reactions stopped at
different times
0
0
10
20
30
40
time (min)
After 25 minutes no additional repair was observed
50
60
70
Results – MLH1 Mutant Endpoint Assay
Question: Given a saturating amount of time, what levels of repair will the mutants exhibit?
2200 bp
1150 bp
1050 bp
L607H
L582V
K571R
R755W
WT
No
MutL
R755W appears to exhibit no repair at all
L607H, L582V, K751R seem to repair about the same as WT
Results – MLH1 Mutant Endpoint Assay continued..
Mutant Endpoint Repair
Negative repair results from
subtraction of negative
control
70
60
50
(unidentified repair pathway)
% repair
40
30
20
10
0
-10
-20
Construct
L607H
L562V
K751R
R755W
WT
Summary
-MutLα is required for repair
-225-250 µg MP1 extract is saturating
-Repair saturates around 25 minutes
-MutLα can be overexpressed in an active form
-Activity can be reliably assayed
-Kinetics are commensurate with previously published data
-L607H, L582V, K751R appear to be capable of wild type levels of repair
-R755W appears to exhibit no activity
Future Studies
-Refining the assay and protein quantitation
-Expand substrate (CT, AG, AC, 8-oxo G, T dimers)
-Different mutants
Thanks to:
Dr. Andrew Buermeyer
Dr. Scott Nelson
Dr. Kevin Ahern
Howard Hughes Medical Institute (HHMI)
Undergraduate Research, Innovation,
Scholarship & Creativity (URISC)