Study of photoinduced electron transfer in fluorescent nucleobase analogues (FBAs) and DNA photolyase

Abstract

Photoinduced electron transfer (PET) plays a crucial role in a wide array of biological pathways. These electron transfer reactions happen from or to the excited state of a chromophore upon absorption of light. Hence understanding the properties of excited states is necessary in elucidating the details of such pathways. The work presented in this thesis deals with PET in two systems: Fluorescent Nucleobase Analogues (FBAs) and DNA photolyase. The introductory chapter (Chapter 1) presents some background information about the two systems and sets up the stage for the reasoning behind the problems addressed in this thesis. FBAs are fluorescent analogues of naturally occurring, weakly fluorescent native nucleic acid bases. When incorporated into single stranded (ss) or double stranded (ds) DNA, the FBA fluorescence is significantly quenched. PET has been implicated to be the cause for the observed quenching. Here we have presented our attempt to correlate the quenching behavior of free FBA: nucleic acid monophosphate (NMP) pairs with the free energies associated with excited state electron transfer delta GET. Based on the delta GET values, we have tried to assign the direction of electron transfer. The quenching behavior of the FBA:NMP pairs were studied through Stern-Volmer (SV) quenching and time-resolved fluorescence studies. The above described analysis has been applied on FBAs: 4-amino-6-methyl-8-(2'-deoxy-beta-D-ribofuranosyl)-7(8H)-pteridone (6MAP), 4-amino - 2, 6 - dimethyl - 8 - (2'-deoxy-beta-d-ribofuranosyl) -7(8H) - pteridone (DMAP), 3-methyl-8-(2'-deoxy-beta-D-ribofuranosyl) isoxanthopterin (3MI) and 6-Methyl-8-(2'-deoxy-β-D-ribofuranosyl) isoxanthopterin (6MI) (Chapter 3), 2-Aminopurine (2AP) (Chapter 4), 8-Vinyl Adenosine (8VA) (Chapter 5). The final part of this thesis (Chapter 6) is on understanding the mechanistic details of a DNA repair process that is due to photoinduced electron transfer in DNA photolyase, a flavoprotein. Before the electron reaches the damaged site in the DNA, the initial electron acceptor in this repair process has been speculated to be the adenine of the flavin adenine dinucleotide (FAD). We have tested this hypothesis by measuring and comparing the various kinetic parameters associated with this process by reconstituting into apo-photolyase the natural cofactor of photolyase (FAD) and an adenine modified flavin (Etheno FAD, epsilon FAD).