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.
| Identifer | oai:union.ndltd.org:uvm.edu/oai:scholarworks.uvm.edu:graddis-2160 |
| Date | 01 January 2019 |
| Creators | Carroll, Brittany |
| Publisher | ScholarWorks @ UVM |
| Source Sets | University of Vermont |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Graduate College Dissertations and Theses |
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