Synthetic approaches to the understanding of DNA nucleobase methylation

DNA methylation is a major source of genetic variation and cancer. Methylation occurs when nucleophilic DNA bases react with methylating agent methyl methanesulfonate (MMS), dimethyl sulfate (DMS), N-methyl-N-nitrosourea (MNU), N-methyl-N-nitro-N-nitrosoguanidine (MNNG), etc. N7-methyl-2'-deoxyguanosine (N7-methyl-dG, or ^{7Me}dG) adduct is the most abundant DNA methylation products for most methylating agents. DNA polymerase actions on ^{7Me}dG are difficult to study due to its instability against ring- opening hydrolysis and deglycosyl ati on. Oligonucleotides containing a single chemical adduct of ^{7Me}dG cannot be chemically synthesized. In addition, ^{7Me}dG is unstable in vivo due to the presence of DNA repair enzymes. This work explores the possibility of using stable analogues of N7-methyl-dG to study the polymerase bypass. The chemical synthesis of N7-methyl-9-deaza- dG (^{7Me9C}dG) has been developed and the nucleoside has been successfully incorporated into oligonucleotides. Thermal melting studies show that replacement of dG by ^{7Me9C}dG only slightly decreases DNA duplex stability. Replication of the DNA templates containing ^{7Me9C}dG and the related 7- methyl -7-deaza- dG (^{7Me7C}dG) and 7-deaza- dG (^{7C}dG) by Klenow fragment of E. coli DNA polymerase I is examined. The misincorporation frequencies on the ^{7Me9C}dG, ^{7Me7C}dG, and 7CdG templates are comparable to the dG template, although the 7-methyl group slows down the turnover rate of the polymerase when dCTP is incorporated. The stability of ^{7Me9C}dG and ^{7Me7C}dG against the actions of formamidopyrimidine DNA N- glycosylase (Fpg) and human alkyladenine DNA Glycosylase (hAAG) are also studied. ^{7Me9C}dG is stable in the presence of both enzymes. In contrast, ^{7Me7C}dG is cleaved by Fpg, and possibly by hAAG but in an extremely slow rate. This work demonstrates that ^{7Me9C}dG is a better analogue than ^{7Me7C}dG for future cellular studies. Epigenetic mechanisms regulate the expression of genetic information. A major epigenetic event is DNA cytosine methylation, which is catalyzed by the DNA methyltransferases (DNMTs). Among different mammalian DNMTs, DNMT1 is the most abundant and active, and plays multiple roles in carcinogenesis, embryonic development, and several other biological functions. Reactivation of silenced tumor suppressor genes by DNMT inhibitors (DNMTi) is provides a relatively new approach to cancer therapy. A couple of irreversible nucleoside DNMT inhibitors have been developed clinically. However, due to their low specificity and high cellular toxicity, there is a clear need for the development of reversible inhibitors. S-adenosyl homocysteine (SAH) is a known strong inhibitor of DNA methyltransferases. Based on the crystal structure of SAH bound to human DNMT1-DNA complex, a series of transition state analogues have been designed to occupy the SAH binding site and the cytosine binding site simultaneously. These analogues have been successfully synthesized from adenosine using a modular approach. Inhibition of DNMT1 by these analogues will be studied in the future.

Name thesis:degree_name

Doctor of Philosophy in Chemistry - (Ph.D.)

Discipline thesis:degree_discipline

Chemistry and Environmental Science

Year

2015

Author dc:creator

• Rana, Jagruti

Contributors dc:contributor

• Haidong Huang
• S. Mitra
• Edgardo Tabion Farinas

• DNA methylation
• Synthesis of 7Me-Deaza-deoxyguanosine
• DNMT
• DNMT inhibitors
• Oligonucleotides
• Polymerase bypass
• Chemistry

Repository record dc:identifier

https://digitalcommons.njit.edu/dissertations/107

OAI identifier oai:identifier

oai:digitalcommons.njit.edu:dissertations-1162