Student Research at Duke

So I think it is finally time to describe my research topic after much procrastination! If I had to sum up my summer project in one sentence, it would be: looking at the role of mismatch repair in suppressing mutations caused by 5-fluorouracil in saccharomyces cerevisiae. But first, a little background information on the mismatch repair pathway and the commonly used antimetabolite 5-fluorouracil. Below is an image that neatly sums up the enzymes and steps involved in this pathway:

Mismatch repair (MMR) is a system that provides various genetic stabilization factors to the cell. Its most well known – and most thoroughly studied – function is to correct DNA biosynthetic errors that arise during DNA replication and recombination. These errors include insertions, deletions, or misincorporation of bases (mismatched bases). Two important genes involved in the mismatch repair system include:

MSH2 – a gene that encodes for a protein necessary for the recognition of errors; when coupled with MSH3 or MSH6 it is capable of recognizing single-base deletion or addition mispairs, larger deletion or addition mispairs, and base-base mismatches

MLH1 – a gene that encodes for a protein that is essential for DNA repair. It joins with PMS2 to form a protein complex that helps coordinate other mismatch repair proteins.

Mutations in either of these essential mismatch repair genes confer with a cancer predisposition syndrome known as hereditary nonpolyposis colorectal cancer (HNPCC). People with HNPCC are at an increased risk of developing colorectal cancer because they have inherited mutations in a key MMR gene that leads to microsatellite instability; they have about an 80% lifetime risk for developing colon cancer. HNPCC is responsible for approximately 2 to 7 percent of all diagnosed cases of colorectal cancer. In total, defects in the MMR pathway (including HNPCC and other non-hereditary defects) are believed to be responsible for the development of about 15% of all colon cancer cases.

Most colon cancer patients are treated with the chemotherapy drug 5-fluorouracil (5-FU for short) which is essentially an analogue of uracil with a fluorine atom in the C-5 position in place of a hydrogen:

5-FU works primarily by inhibiting by inhibiting thymidylate synthase (TS), which is responsible for synthesizing dTMP (thymine) from dump (uracil). Subsequent pyrimidine nucleotide pool imbalances (high concentrations of dUMP and low concentrations of dTMP) eventually causes high levels of uracil incorporation into DNA. Although no one is exactly sure why uracil incorporation causes cell toxicity, it has been suggested that futile cycles of uracil excision and repair finally lead to cell cycle arrest and apoptosis.

My mentor, Sally York, believes that another effect of nucleotide pool imbalances and incorporation of FdUMP (5-FU) into DNA is that the likelihood of mutation during replication is higher (abnormal levels of adenine, thymine, guanine, and cytosine makes it more likely that DNA polymerase will not have access to the base it needs during replication, and thus more likely that it will make a mistake). Normally, mismatch repair is able to suppress these mutations by recognizing and excising any errors. However, in colon cancer patients who are mismatch-repair deficient (remember I mentioned that this is about 15% of all colon cancer patients), any errors in replication are not fixed because the mismatch repair pathway is dysfunctional. This has significant implications because tumor cells of MMR-deficient patients will actually be more likely to develop mutations when exposed to the drug 5-FU. A higher mutation makes it probable that a tumor cell will develop resistance to further treatment.

For my research project, I will be trying to answer the following question: What role does MMR play in cellular processing in the presence of 5-FU? Is MMR involved in suppressing mutations brought about by incorporation of FdUMP into DNA? If this is true, MMR-deficient cells should show an increased mutation rate in the presence of 5-FU. In order to answer this question, I will be analyzing the mutation rates, mutational spectra, and survival curves of various yeast strains that are deficient in a specific gene of the MMR pathway to ascertain the role of that gene in suppressing mutations brought about by 5-FU. I will be using the yeast saccharomyces cerevisiae because they are easy to manipulate genetically, so they are ideal organisms for mutator and mutation spectrum analyses (also, the MMR system is generally conserved among eukaryotes).