Functional consequences Functional consequences of NLS mutations in of NLS mutations in

human MLH1human MLH1Alex DukesAlex Dukes

Dr. Andrew BuermeyerDr. Andrew BuermeyerDepartment of Environmental & Department of Environmental &

Molecular ToxicologyMolecular ToxicologyOregon State UniversityOregon State University

HHMI Program, Summer 2007HHMI Program, Summer 2007

The Importance of The Importance of Mismatch Repair (MMR)Mismatch Repair (MMR)

Cellular mechanism for preventing DNA mutations, resulting from:Cellular mechanism for preventing DNA mutations, resulting from: Replication errorsReplication errors Recombination pathwaysRecombination pathways Exogenous DNA damaging agentsExogenous DNA damaging agents

Necessary to trigger apoptosis in event of extensive DNA damageNecessary to trigger apoptosis in event of extensive DNA damage

MMR and Colon CancerMMR and Colon Cancer

Colorectal cancers Colorectal cancers account for 10% of all account for 10% of all new cancers new cancers

Lynch Syndrome, a Lynch Syndrome, a type of hereditary type of hereditary colon cancer (HNPCC)colon cancer (HNPCC)

Caused by genetic Caused by genetic defects in MMR genesdefects in MMR genes

©2005 American Cancer Society, National Cancer Institute

A Component of MMR: A Component of MMR: MutLMutLαα

Heterodimer composed of MLH1 Heterodimer composed of MLH1 and PMS2 proteinsand PMS2 proteins

In the absence of MMR, cells In the absence of MMR, cells display:display: Increased mutation rate Increased mutation rate Decreased apoptotic response to Decreased apoptotic response to

genotoxins genotoxins Mutations in MLH1 account for 30-Mutations in MLH1 account for 30-

40% of Lynch Syndrome cases40% of Lynch Syndrome cases

Two Mutations of Two Mutations of InterestInterest

Missense mutations both identified in cancer Missense mutations both identified in cancer patientspatients

Nuclear localization sequence (NLS): Nuclear localization sequence (NLS): Acts as a cellular “password” to allow protein into Acts as a cellular “password” to allow protein into

nucleus via the nuclear porenucleus via the nuclear pore amino acids 470 – 474 in MLH1amino acids 470 – 474 in MLH1

Image courtesy of : www.scripps.edu/.../proj/NPC/NucleusWhite.gif

Image courtesy of: Wu, X., et al, Dimerization of MLH1 and PMS2 Limits Nuclear Localization of MutL. Molecular and Cellular Biology, 2003. 23(9): p. 3320–3328

Mutation #1Mutation #1 Amino acid 474Amino acid 474 Replaces arginine (R) with Replaces arginine (R) with

glutamine (Q)glutamine (Q) Charged Charged Charged Charged

R474QR474Q

Images courtesy of: www.contexo.info/.../images/aminoacidsweb.gif

Mutation #2Mutation #2

Amino acid 472Amino acid 472Replaces arginine (R) with isoleucine Replaces arginine (R) with isoleucine (I)(I)Charged Charged Non-polar (more Non-polar (more deleterious?)deleterious?)

R472IR472I

Images courtesy of: www.contexo.info/.../images/aminoacidsweb.gif

Project ObjectivesProject Objectives Examine two NLS mutations in MLH1 and Examine two NLS mutations in MLH1 and

identify the phenotypic consequences of identify the phenotypic consequences of eacheach

Experimental MethodsExperimental Methods Transient transfectionTransient transfection Stable transfectionStable transfection

The Transient The Transient TransfectionTransfection

Plasmids encoding mutated MLH1 and PMS2 Plasmids encoding mutated MLH1 and PMS2 are introduced into MutLare introduced into MutLαα- deficient cells- deficient cells

Cells assessed to determine protein Cells assessed to determine protein expression and localization patternexpression and localization pattern

Is the mutant MutLIs the mutant MutLαα heterodimer stable? heterodimer stable?Does the mutation interfere with normal localization patterns?Does the mutation interfere with normal localization patterns?

Nuclear Cytoplasmic

MutLα present

MutLα absent

The Transient The Transient TransfectionTransfection

Lipid-based transfection

The Stable TransfectionThe Stable Transfection

Plasmid encoding mutated MLH1 is Plasmid encoding mutated MLH1 is introduced into MLH1- deficient cellsintroduced into MLH1- deficient cells

Cells exhibiting reduced MMR Cells exhibiting reduced MMR function identified by:function identified by: Increased mutation levels Increased mutation levels Loss of apoptotic responseLoss of apoptotic response

Does the mutation result in reduced MMR function? Does the mutation result in reduced MMR function?

This assay also can be used to confirm the This assay also can be used to confirm the heterodimer stability and localization pattern heterodimer stability and localization pattern

found in the transient transfection.found in the transient transfection.

The Stable TransfectionThe Stable Transfection

Selection with cytotoxic G418

Positive ControlNegative Control R472I R474Q

Electroporation

Plating from the Stable Plating from the Stable TransfectionTransfection

Freeze cell lines

Ouabain AssayMutation frequency Survival following

DNA damage response

6-TG Assay

Pass 1:10

Pass 1:10

Interpretation of possible Interpretation of possible outcomesoutcomes

LocalizationLocalization MMR MMR PhenotypePhenotype

Interpretation of ResultsInterpretation of Results

CytoplasmicCytoplasmic DeficientDeficientMutation interferes with Mutation interferes with localization causing MMR localization causing MMR deficiencydeficiency

Hypothesis: Both mutations interfere with Hypothesis: Both mutations interfere with localization and cause MMR deficiency.localization and cause MMR deficiency.

CytoplasmicCytoplasmic ProficientProficient Low levels of MutLLow levels of MutLαα enter enter nucleus and are sufficient for nucleus and are sufficient for MMR phenotypeMMR phenotype

NuclearNuclear DeficientDeficient

NuclearNuclear ProficientProficient

Mutation interferes with Mutation interferes with enzymatic function or other enzymatic function or other protein activity, not localizationprotein activity, not localizationMutation does not interfere Mutation does not interfere with normal localizationwith normal localization

Transient Transfection Transient Transfection ResultsResults

PMS2

Only

R474Q

R47

2I

1 2 3 4W

ild T

ype

MSH6

PMS2

MLH1

Both R474Q and R472I mutants stabilize PMS2

Stable Transfection Stable Transfection Results: Results:

Protein Expression LevelsProtein Expression Levels

MLH1 accumulates similar to wild type

MLH1 stabilizes PMS2 similar to wild

type

Stable Transfection Stable Transfection Results: Results:

Cellular LocalizationCellular Localization

Mutation #2: R472IMutation #1: R474Q

Both mutants exhibit nuclear localization

Stable Transfection Stable Transfection Results: Results: Mutation FrequencyMutation Frequency

MMR Proficient

(LOW mutant frequency)

MMR Deficient

(HIGH mutant frequency)

Stable Transfection Stable Transfection Results: Results: Cytotoxic ResponseCytotoxic Response

MMR Proficient

(LOW survival)

MMR Deficient (HIGH

survival)

Summary of ResultsSummary of Results Localization?Localization? NUCLEAR

PROFICIENT Mismatch repair phenotype?Mismatch repair phenotype?

Possible ExplanationsPossible Explanations

A redundant NLS sequence allowed the protein into the A redundant NLS sequence allowed the protein into the nucleus (Another in MLH1? In PMS2?) nucleus (Another in MLH1? In PMS2?)

1

2

The mutation in MLH1 did not critically interfere with the NLSThe mutation in MLH1 did not critically interfere with the NLS

Colon Cancer Colon Cancer ImplicationsImplications

Our functional assays show no Our functional assays show no reduced MMR activityreduced MMR activity

Other lab groups obtained similar Other lab groups obtained similar results for R474Qresults for R474Q

Clinical data is not conclusive in Clinical data is not conclusive in linking mutations to Lynch Syndromelinking mutations to Lynch Syndrome

CONCLUSIONSCONCLUSIONS1.1. The mutations are not pathogenic.The mutations are not pathogenic.2.2. The mutations are moderately The mutations are moderately

pathogenic and our assays are not pathogenic and our assays are not sensitive enough.sensitive enough.

AcknowledgementsAcknowledgements

Howard Hughes Medical InstituteHoward Hughes Medical Institute Dr. Andrew Buermeyer, mentorDr. Andrew Buermeyer, mentor Dr. Kevin Ahern, program coordinatorDr. Kevin Ahern, program coordinator

Generously funded by grants Generously funded by grants from the HHMI Programfrom the HHMI Program