Exploring DNA damage and Repair through Molecular Dynamics Simulations

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Campbell, Arthur John
The Graduate School, Stony Brook University: Stony Brook, NY.
Everyday our cells are exposed to various forms of endogenous and exogenous DNA damaging agents. One of the most cytotoxic forms of endogenous damage comes from Reactive Oxygen Species. Oxidative DNA damage has been linked to a number of human diseases, including cancer. The most common oxidative lesion found in DNA is 8-oxo-guanine, or 8-oxo-G. It is known that formamidopyrimidine DNA glycosylase (Fpg) distinguishes between undamaged guanine bases and damaged 8-oxo-G, however, the mechanism by which this discrimination is carried out is currently under debate. There are a number of X-ray crystallographic structures available that highlight Fpg's damage recognition and base eversion process that involves the repair of these lesions, yet there are some limitations to these experiments. These experimental structures are only snapshots along the reaction coordinate, therefore more detail is needed to understand Fpg's mechanism between these snapshots. In this work, computer modeling was used to measure the eversion process as well as pinpoint key components involved in damage recognition of 8-oxo-G. A form of exogenous damage of particular interest in this work is the cancer chemotherapeutic agent Nitrogen Mustard (NM). NM is known to form various crosslinks in DNA. The crosslink that has been shown to be the most effective in inhibiting cell growth and facilitating apoptosis is the 1,3 interstrand crosslink formed in a 5' GpNpC sequence. Little structural information is known about this crosslink because it is difficult to study experimentally, as it is a promiscuous agent forming various types of crosslinks that are prone to depurination. The SchÇÏrer laboratory has created a protocol to synthesizing a stable analog of NM but it is unknown if this analog is a good mimic of the native NM adduct. In this work, molecular modeling was used to compare the natural NM to its chemically stable analog to validate the use of these analogs. These crosslinks were also compared to undamaged DNA to quantify the amount of distortion created by a 1,3 NM crosslink.