Q & A with Christopher J. Wilds, MCIC: Canada research chair in biological chemistry.
Q: How would you describe your research?
In a nutshell, my research utilizes organic chemistry to assemble novel DNA molecules that are used to probe various biochemical processes such as HIV-1 replication and resistance of cancer cells to chemotherapy.
One such process that we are currently interested in is DNA repair. Some of the clinically-used chemotherapeutic agents, namely bifunctional alkylating agents, act upon the DNA in cancer cells by basically locking the two strands together. This in turn prevents the DNA strands from coming apart, thus inhibiting replication and ultimately the cancer cells die. But, as it turns out, some people develop a resistance to these therapies because the cells are capable of reversing this damage. As a result DNA replication continues and cancer progression persists.
Q: Can you explain the main thrust of your research team?
The main goal of this project is to understand the mechanisms by which cells recognize and repair the damages (desired) induced by current chemotherapies, which makes them ineffective as drugs. We first prepare interstrand cross-links that mimic what lesion the drug would introduce in the cell between two single strands of DNA via organic synthesis. The construction of these lesions, starting with the organic synthesis of nucleoside dimers, forms one major focus of my research. We then assemble short DNA duplexes incorporating these novel lesions utilizing an instrument called a DNA synthesizer. This is followed by investigating the structural changes introduced by these lesions on the duplex. These structural changes could serve as a signal to the repair enzymes that this is a site where there is damage. Finally, we use DNA repair enzymes to examine whether these enzymes are capable of reversing the damage.
Q: What kind of insights have you made?
We have successfully constructed DNA duplexes containing interstrand alkyl cross-links between the O6 atoms of 2'-deoxyguanosines and have shown that a repair protein is able to repair the duplex when the alkyl chain is 7 carbons long but not when the length is reduced to 4. The latter is very stable and we propose that the protein's access to the damage site is limited. Using this observation, ideally if one is to inhibit the repair protein in question with such DNA molecules either alone or in combination with current therapies, this would serve to increase the effectiveness of the current drugs used in the combat of cancer.
The understanding of the processes of recognition and repair of DNA lesions by the repair machinery is a multi-team effort involving several research disciplines such as organic synthesis, biochemistry and enzymology.
Q: What are the specific things which take place in your lab at Concordia?
Currently, we have the capacity to synthesize mimics of clinically relevant interstrand cross-links. Of the several novel dimers we have prepared to date, one of them is an analogue of a cross-link that is formed by hepsulfam. Simply stated we connect the two heterocyclic bases of DNA specifically at the O6 atoms of two guanines with an alkyl linker using a procedure known as the Mitsunobu reaction. We can construct symmetrical or non-symmetrical dimers depending on the orientation of the linkage we wish to introduce in our cross-linked DNA molecule.
Once we have made this dimer building block we incorporate it into a DNA duplex via solid-phase oligonucleotide synthesis using a DNA synthesizer which allows us to assemble relatively small DNA molecules in a sequence specific manner. DNA synthesizers have been used for over two decades by several research groups who introduce various modifications into DNA for a number of purposes including antisense, antigene and diagnostic applications. We have taken this one step further by synthesizing complete duplexes that contain modifications that happen to contain cross-linked DNA bases. We use a number of techniques such as polyacrylamide gel electrophoresis (PAGE) and high performance liquid chromatography (HPLC) to purify these cross-linked duplexes for DNA repair studies as well as structural studies such as nuclear magnetic resonance (NMR) and X-ray crystallography.
Q: Besides synthesis, what else do you do with these substrates?
Although primarily synthetic, my research over the last two years has progressed to studies investigating direct repair of our cross-linked DNA molecules here at Concordia where I co-supervise a student with my colleague Judith Kornblatt. We are capable of studying direct repair of DNA containing single O6 adducts as well as the cross-linked duplexes by E. coli repair proteins. Preliminary biophysical and structural work such as duplex stability and investigation of structural deformation of the DNA induced by these lesions are also conducted in my lab at Concordia. It is my hope that we can determine the structures of these cross-linked duplexes with repair proteins in the near future.
Q: What have you found?
We have recently demonstrated in collaboration with Anthony Pegg (Penn State University) for the first time that human alkyl guanine transferase can repair interstrand cross-linked DNA duplexes containing an alkyl linker of 7 methylene groups, whereas the protein is not able to repair a 4 carbon cross-link, and this work was recently published in the journal Biochemistry. The enzyme, alkyl guanine transferase, specifically reverses alkylation damage at the O6 atom of guanine. It is known that this enzyme can remove relatively small alkyl substituents at the O6 position. We showed for the first time that an O6-2'deoxyguanosine alkyl interstrand cross-link could be repaired by this particular enzyme. It is a substrate that would be relatively challenging to repair because the alkyl chain connects the two strands together and for the enzyme to repair it, the damaged base has to be flipped out of the duplex into the enzyme's active site. The thing about our cross-linked DNA is that there is a lot of cargo at the damaged site since it has the other strand attached to it. To us it is a very interesting finding because it is an enzyme that has not been shown to directly repair interstrand cross-links before and this is the first report of it being able to do so.
Q: What are you working towards?
We are examining a number of related substrates that can evade repair by these DNA repair enzymes. One option is to directly inhibit the enzymes that reverse damage so that the existing therapies can remain and continue to be effective via a combination therapy. The other more challenging path is to design new drugs that will not be recognized and repaired by the repair machinery. It is hoped that both routes will slow down the progression of cancer, which is a multi-team effort both nationally and internationally.
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|Publication:||Canadian Chemical News|
|Date:||Nov 1, 2008|
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