Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)


Cellular, Molecular and Biomedical Sciences

First Advisor

Sylvie Doublié


Cells synthesize proteins, the molecular instruments of all cellular processes, via

intermediate biomolecules that are susceptible to damage at every step. Known as the

central dogma of molecular biology, genes encoded in deoxyribonucleic acid (DNA) are

transcribed, spliced, and matured into messenger ribonucleic acid (mRNA). These

nucleic acids direct protein synthesis by the pairing of nucleotide triplets with transfer

RNA (tRNA). tRNAs concomitantly decode the so-called codon, as they escort the

correct amino acid to the ribosome for extension of the nascent polypeptide chain.

Damage to any of these intermediate biomolecules can be highly damaging to protein

synthesis, leading to aberrant biochemical processes, aging, cancer, or apoptosis.

Accordingly, cells have evolved essential response and repair pathways to ensure that

replication, transcription, and translation occur with high fidelity. In this dissertation, we

interrogate two enzymes involved in these quality-control measures: 1) a DNA

glycosylase which recognizes damage to the DNA bases, and 2) a tRNAHis

guanylyltransferase-like protein (or THG1-like proteins, TLPs) which repairs truncated or

mismatched tRNA via 3’5’ polymerization.

DNA is assaulted daily to the tune of 30,000 lesions per cell per day by

exogenous and endogenous stressors. One of many DNA repair pathways, the base

excision repair (BER) pathway, removes the small non-bulky, and oxidized DNA lesions

from the genome. DNA glycosylases are the first enzymes in the concerted mechanism

tasked with scanning the entire genome for DNA damage and initiating the repair of

lesions. The human genome encodes 11 DNA glycosylases, which possess overlapping

substrate specificities within BER. The DNA glycosylase, endonuclease three (Nth),

recognizes and removes oxidized pyrimidines during all phases of the cell cycle. We have

solved the first X-ray crystal structure of human Nth-Like 1 (hNTHL1), which revealed a

novel open conformation. This unprecedented example of an Nth DNA glycosylase

undergoing interdomain rearrangement provides important insight into the molecular

mechanism of this critical guardian of the genome.

In eukaryotes, tRNAs must be modified at the 5’ end during maturation. tRNAHis

guanylyltransferase (THG1), an essential gene in yeast, catalyzes the addition of guanine

to the 5’ end of tRNAHis. Reverse polymerization requires adenylation (or guanylation) to

activate the 5’ end of the tRNA. After adenylation, there is a shift of the 5’-phosphate of

the tRNA to accommodate the forthcoming nucleophilic attack by the 3’-OH of the

incoming nucleotide. In contrast to their human counterparts, the archaeal TLP enzymes

utilize the 3’ to 5’ NTP-polymerization reaction to repair 5’-degraded tRNA molecules.

We have solved the first crystal structure of a TLP caught in an intermediate step

following activation by guanylation, showing that the base rotates within the nucleotide

binding site to align the active site.



Number of Pages

194 p.

Included in

Biochemistry Commons