Biochemical Defects Associated with Cancer-Causing PathogenicMutations in Human MLH1

Andrew Nguyen

Laboratory of Dr. Andrew BuermeyerDepartment of Environmental and Molecular Toxicology

Oregon State University

©2005 American Cancer Society, National Cancer Institute

Deaths Caused by Cancer – United States, 2005

Approximately 56,000 deaths fromcolorectal cancer in 2005 alone

Women 273,560 deaths

Colon and rectum 10%

Men 291,270 deaths

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 DNAPolymerase

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 ina condition known as Lynch Syndrome. This disorder is characterized by a predispositionto early onset of colorectal and other internal cancers.

Knowing who is at risk for cancer because of MMR deficiency can influence treatmentoptions for existing patients or surveillance strategies for higher risk people.

DNA Mismatch Repair (MMR)

MMR acts to maintain genomic integrity by correcting DNA replication errors

Evolutionarily conserved process

Mechanism involves several protein dimersnecessary for successful repair

MutLα is composed of two separate proteins,MLH1 and PMS2

Mechanism of Mismatch Repair

Recognition

Excision

Resynthesis

G

T

T

T

A

MutS

MutLExonuclease

DNA polymerase

In-Vitro strand choice is determinedby nick in non-template strand

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 siteon the plasmid

MMR activity assessed by percentage of plasmid substrate that can be restricted

Resynthesis Restriction

Ban1 restriction siteBan1 restriction site

Excision

% repair

0 50 100

Ban1 restriction site

Creation of Mismatch Containing Plasmid Substrate

pRO1

Start with the pRO1 plasmid

Nick plasmid

Generate gap by adding excess ofcomplementary oligonucleotide

Anneal a DNA fragment of knownsequence that will create a mismatch

Mismatch substrate

Experimental Approach – MP1 titration

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

Increasing amounts of MP1 added

0NoMutL

Repair efficiency CT loop

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

0 50 100 150 200 250 300 350

ug mp1

% repair

pk height

Repair saturated around 225-250 µgRepair is MutL, MP1 dependent

Question: How much non-MutL containing cell extract is saturating?

MLH1 WT Kinetics

0

10

20

30

40

50

60

70

80

0 10 20 30 40 50 60 70

time (min)

% repair

Time points

Experimental Approach – MLH1 WT Kinetics

After 25 minutes no additional repair was observed

Identical repair reactions stopped atdifferent times

Minutes0 5 10 15 20 25 30 35 40 45 50 55 60

Question: How does repair progress with time?

Results – MLH1 Mutant Endpoint Assay

R755W appears to exhibit no repair at all

L607H, L582V, K751R seem to repair about the same as WT

2200 bp

1150 bp

1050 bp

L607H L582V K571R R755W WTNoMutL

Question: Given a saturating amount of time, what levels of repair will the mutants exhibit?

Results – MLH1 Mutant Endpoint Assay continued..

L607H L562V K751R R755W WT

Mutant Endpoint Repair

-20

-10

0

10

20

30

40

50

60

70

Construct

% repair

Negative repair results fromsubtraction of negative

control

(unidentified repair pathway)

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)