Maintaining genomic integrity is essential for cellular viability. Alteration of bases can occur via exposure to reactive oxygen species. There exist cellular repair mechanisms to correct aberrant bases which can lead to single strand breaks. The primary repair mechanism the cell employs for damaged bases is Base Excision Repair (BER).
The steps of BER involve identifying and removing the damaged base with a DNA glycosylase, incising the DNA backbone and processing the DNA ends by an endonuclease, filling the abasic site by DNA polymerase, and sealing the DNA backbone by DNA ligase. While the steps of BER have been well characterized, there is still much to investigate about how BER is regulated. Studies have suggested that BER proteins undergo post-translational modification. Additionally, proteins not previously shown to participate in BER are being recognized as regulating BER through protein localization, turnover, or interactions.
The purpose of this thesis was to identify and characterize novel modulators of BER proteins and activity. The first modulator described is β1 integrin engagement. It was previously described that integrin engagement can reduce 3’OH and phospho-H2A.X formation. In Chapter 2, the effects of β1 integrin-engagement on reducing total DNA breaks in murine lung endothelial cells (MLECs) will be discussed. β1 integrin-engagement did not reduce the total number of breaks in DNA after genotoxic insult, therefore the effect of integrin-engagement on processing of DNA breaks was examined. Apurinic/Apyrimidinic Endonuclease 1 (APE1) is the primary endonuclease responsible for incising the DNA at an abasic site. In Chapter 3, the effect of integrin-engagement on APE1 activity and interaction with X-Ray Cross Complementing Repair Protein 1 (XRCC1) will be discussed. APE1 and XRCC1’s interaction stimulates APE1 activity. Integrin-engagement in the presence of H2O2-induced damage reduced APE1-XRCC1 interaction and APE1 activity. This provides a potential explanation for the reduced 3’OH previously observed after integrin-engagement.
The second modulator of BER that was identified is Protein Never in Mitosis Gene A Interacting-1 (PIN1). PIN1 is the only peptidyl-prolyl isomerase that is capable of recognizing a phosphorylated serine or threonine followed by a proline and isomerizing the prolyl bond between the cis and trans conformations. In Chapter 4, two putative PIN1 binding sites were identified in APE1 and it was found that PIN1 and APE1 interact. Furthermore, it was shown that threonine 232 in APE1 is necessary for this interaction. In Chapter 5, the effect of knocking down (KD) PIN1 protein levels on APE1 function is examined. PIN1 KD does not alter the levels of APE1 protein or APE1 protein localization in murine mammary adenocarcimoma cells (EMT6). APE1 interaction with XRCC1 was increased in PIN1 KD cells as was APE1 activity.
In Chapter 6, the effects of β1 integrin engagement on APE1 as well as the role PIN1 status plays in APE1 function are summarized. The more that is understood about regulation of BER proteins, the more effective intervention can be designed to help prevent and treat disease. The data presented here suggest that APE1 can be regulated through extracellular (integrin-engagement) and intracellular (PIN1) processes.