Research in the Spratt lab addresses the fundamental question of how cells deal with DNA damage. The long-term objective of this project is to understand the chemical interactions that govern the high fidelity replication of DNA.
Mechanisms of fidelity and mutagenesis of DNA polymerases.
Out most recent project involves identifying the mechanism by which O2-POB-dT, a DNA adduct derived from tobacco smoke, causes mutations. Our approach will involve both in vitro enzymology and cell culture work in identifying the critical proteins involved in the processes.
DNA repair and carcinogenesis.
Deficient DNA repair is found to be associated with an increased risk of cancers at the population level. However, the assays used are ill-suited to evaluate an individual’s risk. We are investigating assays using highly specific HPLC-MS/MS and next generation sequencing technology to develop new assays that we aim to evaluate an individual’s susceptibility to specific cancers.
Techniques we use:
- Organic Synthesis
- We synthesize molecules to probe mechanisms of polymerase activity.
- Enzyme kinetics
- With transient – state enzyme kinetics, we explore intermediates along the reaction mechanism.
- Next generation sequencing
- We use NGS to identify position in the genome that are damaged , and locations of polymerase activity.
Organic synthesis
3-deaza-2′-deoxyguanosine is used to examine the interactions between the minor groove of the DNA and the polymerase. We found that these interactions are crucial for catalysis and fidelity of A-family DNA polymerases
O²-(4-(3-pyridyl)-4-oxobutyl)-thymidine was synthesized to examine if this adduct derived from cigarette smoking was mutagenic. We found that during DNA replication, both dA and dT are inserted opposite this adduct. In addition we found that DNA polymerases eta, zeta and REV1 are involved in the bypass in cells
N²-(4-Ethynylbenzyl)-deoxyguanosine 5′-triphosphosphate reacts rapidly with DNA polymerase κ, but not with other polymerases. We will use the nucleoside as a reagent to probe the activity of pol κ in cells.
Enzyme Kinetics
Using transient state kinetic analysis we showed that DNA polymerase nu catalyses both correct and incorrect DNA synthesis rapidly. Previously, it was thought that low fidelity polymerases are low fidelity because they are inefficient at catalyzing correct base pair formation. Here we show the opposite.
Next Generation Sequencing
Utilized N²-(4-Ethynylbenzyl)-deoxyguanosine to probe the sequence-specific activity of DNA polymerase κ
