Structural studies of two proteins involved in the maintenance of genomic stability FEN 1 and DNA-PKcs

Abstract

Genomic stability refers to an organism’s ability to maintain and pass forward its genetic information. There are a raft of proteins and pathways whose sole purpose is maintaining this stability through swiftly replicating DNA as well as accurately repairing damage caused through contact with endogenous and exogenous DNA damaging elements.

This study will focus on the structural aspects of two proteins that play a part in different areas of genome maintenance. Flap Endonuclease 1 (FEN 1) works in DNA replication, where it is tasked with removing a small RNA flap that is created during Okazaki fragment formation. This flap removal is essential to mature these fragments into one continuous strand of nascent DNA.

Using the archeon Pyrococcus abyssi (Pab) as a model system has the advantage of possessing simple replicative machinery, whilst bearing striking similarities with the human system. Pab is a hyperthermophilic, piezophile meaning it thrives in conditions of high temperature and pressure.

DNA-dependent protein kinase (DNA-PK) is a holoenzyme that plays a role in the Non Homologous End Joining (NHEJ) pathway by repairing DNA double strand breaks (DSB’s). In cancer therapy, a patient is exposed to DNA damaging elements, leading to an ever-increasing population of DSBs. If an inhibitor of DNA-PKcs were introduced along with this therapy it could potentiate its effect, as the cancerous cells will be less able to repair the damage. The aim of this part of the study is to determine a protocol to generate pure, soluble, correctly folded protein for the purposes of biophysical characterisation and X-ray crystallographic structural studies.