PhD dissertation_Ritu Chaturvedi.pdf (34.66 MB)
Download file

Reactivities Leading to Potential Chemical Repair of Sunlight-Induced DNA Damage: Mechanistic Studies of Cyclobutane Pyrimidine Dimer (CPD) Lesions under Alkaline Conditions

Download (34.66 MB)
thesis
posted on 07.01.2021, 15:08 by Ritu ChaturvediRitu Chaturvedi

Cyclobutane pyrimidine dimers (CPD) are the predominant DNA lesions formed upon exposure of this biopolymer to sunlight. Given the potentially dire biological consequences of DNA lesions, there is a need to fully characterize their behaviour, with an eye towards understanding their complete reactivity and as a possible means to detect and quantify their presence in the genome. The work described in this dissertation describes studies of the alkaline reactivity of CPD lesions generated within dinucleotide & polynucleotide strands. It was found that CPD-TpT is generally inert under alkaline conditions at room temperature, which is in agreement with earlier studies on alkaline hydrolysis of CPD-thymine and CPD-thymidine. However, a re-evaluation of the same reaction in the presence of 18O labelled water demonstrated that, similar to other UV-induced DNA lesions containing a saturated pyrimidine ring, CPD undergoes a water addition at the C4=O group of the nucleobase leading to the formation of a hemiaminal intermediate. This intermediate, however, does not lead to hydrolysis products and completely reverts to starting material under those same conditions. Moreover, the two C4=O groups present on 3′ and 5′-thymines in a CPD molecule show different chemical reactivities, with the 3′ C4=O group having greater affinity towards water addition as compared to the one on 5′ end, a fact reflected in different rates of exchange with the incoming nucleophile leading to the hemiaminal intermediate. The 18O labelling reaction was also investigated in CPD lesions generated within oligonucleotides to probe the cause of asymmetry between the 3′ vs 5′ C4=O groups; ultimately, it was determined that the asymmetric reactivity observed to occur between the two C4=O groups was an intrinsic property of the CPD molecule and did not arise as a result of asymmetry in a dinucleotide setting.


In addition to the above studies, during the course of the investigation of the nucleophilic reactivity of CPD, a chemical reaction was observed leading to what appeared to be the rapid and total chemical reversal of CPD lesions to the original TpT (thymine-thymine dinucleotide)! This “repair” reaction occurred when CPD reacted with hydrazine, and appears facilitated by an inert atmosphere under which it rapidly proceeds to completion at room temperature.


History

Degree Type

Doctor of Philosophy

Department

Chemistry

Campus location

Indianapolis

Advisor/Supervisor/Committee Chair

Dr. Eric C. Long

Additional Committee Member 2

Dr. Michael J. McLeish

Additional Committee Member 3

Dr. Jingzhi Pu

Additional Committee Member 4

Dr. Ian K. Webb

Usage metrics

Licence

Exports