Kinetics of MutSα-Mediated DNA Mismatch Repair

William GibsonDr. John Hays

Oregon State UniversitySummer 2010

The Importance of DNA Mismatch Repair (MMR)

A poorly working MMR system can cause many problems such as accelerated cell aging, cell death, and cancer.

Diseases caused by defects in MMR systems are costly and damaging because of their severity and the lack of treatment options available.

MutSα-MMR System

MutSα-MMR system 3 main components

MutSα – Two subunits which form a dimer to bind DNA mismatches or adducts.

MutLα – Two subunits which assist MutSα and exhibit a nuclease function.

PCNA – A trimeric clamp which helps guide MutSα and MutLα along the DNA strand.

Mut-LαMut-Sα

PCNA

DNA Damage from PAH Alkylation

Polycyclic aromatic hydrocarbons (PAHs) attach to DNA and cause lesions.

PAH's are produced by the combustion of complex carbon compounds (ex. coal).

What I Want to Do

• Examine MMR initiation at gaps vs. nicks.• Examine how the MMR system responds to adducts

such as a PAH-guanine (PAH-G) adduct.• Determine if PAH-G mismatches provoke MMR

excision similar to standard base mismatches.

Plasmid Model for MMR• Initiation: MutSα complexes with MutLα and PCNA.

• Proximal Excision: MutSα-MMR complex excises to the mismatch.

• Distal Excision: MutSα-MMR complex excises past mismatch before dissociating.

Plasmid Construct

The plasmid construct has multiple nicking and gapping sites to initiate repair.

PAH-Guanine is opposite to any of the 4 bases (A, G, T, C)

Experimental DesignGrowth of Plasmid

↓Addition of Gap and Mismatch Sites

↓MMR Assay (Using Radio-Labeled Reporter

Oligos)↓

Results Analysis (Compare Gapped vs. Nicked and 3 Different Mismatches vs.

Controls)

Growth of Plasmid

Addition of Gap and

Mismatch SitesMMR Assays Results

Analysis

Growth of Plasmid

• The plasmid is modified from the highly used pUC19 plasmid.

• The plasmid contains a drug resistance gene.

Growth of Plasmid

Addition of Gap and

Mismatch SitesMMR Assays Results

Analysis

Gapping and Nicking

• Multiple unique restriction sites allow different areas to be targeted.

• Modified restriction enzymes allow nicking of a single strand at nick sites.

Growth of Plasmid

Addition of Gap and

Mismatch SitesMMR Assays Results

Analysis

MMR Assay 1

• Excision Assay: Measure Gapped vs. Nicked initiation.

Repair No Repair

Growth of Plasmid

Addition of Gap and

Mismatch SitesMMR Assays Results

Analysis

MMR Assay 1No Repair Repair Time 0 Time 2 Time 4 Time 6

Growth of Plasmid

Addition of Gap and

Mismatch SitesMMR Assays Results

Analysis

MMR Assay 2• PAH Assay: Measure relative abundance of the radiolabeled probes.

Excision past PAH

Excision but not past PAH

No excision triggered

Growth of Plasmid

Addition of Gap and

Mismatch SitesMMR Assays Results

Analysis

Results Still Pending

• The first assay will provide a definitive answer on gap vs. nick initiation, which will allow future assays to use the most efficient initiation method.

• By exploring the behavior of the MMR system in regards to PAH-G adducts, specific kinetic models can be built to further understand the MMR process at a molecular level.

Acknowledgments

Dr. John Hays and Peter Hoffman

Colin Tominey

Dr. Mark Curtis, Johanna Steinbrecher, Robert Ursu, Ana Brar, Taylor Hoffman, Buck Wilcox

Figures: ATSDR (slide 2), JBC (slide 4), myself (slide 3,6,7,11,12,13), NEBcutter (slide 10), David Brooks (slide 9)

Special thanks to Kevin Ahern and the HHMI program for letting me take part!