Ribonucleotide reductase is a remarkable enzyme that catalyzes the rate-limiting
step in the synthesis of the 2'-deoxynucleoside triphosphates. The intent of this project
was to characterize the ribonucleotide reductase encoded by the orthopoxvirus,
vaccinia. The first objective was to study the structural and functional features of the
viral small subunit...
This dissertation describes an investigation of DNA precursor supply for mitochondrial and nuclear DNA synthesis in HeLa cells and Mouse L-cells. HeLa cells were used for the quantitation of cellular and mitochondrial deoxyribonucleoside triphosphate (dNTP) and ribonucleoside triphosphate (rNTP) pools and of changes in pools in response to treatment with...
Ribonucleotide reductase is an important enzyme in the control of
DNA replication within the cell. Ribonucleotide reductase exerts its
control through enzymatic reduction of nucleoside diphosphates. The
bacteriophage T4 enzyme is an example of the class of iron-requiring
reductases which also includes E. coli and mammalian ribonucleotide
reductases. The two...
The dihydrofolate reductase gene of bacteriophate T4
has been investigated at the structural level to elucide the
unusual regulation of expression of this gene and possible
evolutionary relationships between the ohage and drug resistance
factors-encoded dihydrofolate reductases (DHFR).
A 1.1-kilobase-pair restriction fragment of the T4
multiple mutant dec8 containing frd,...
Investigators have been studying an aggregate
containing enzymes of deoxyribonucleoside triphosphate
biosynthesis in T4-phage infected bacteria. They suggest
that it behaves as an organized complex, efficiently
channeling DNA precursors to the replication apparatus.
Previous work has suggested that about 10 enzyme
activities remain associated through several fractionation
steps. This multi-enzyme...
A rapid nuclear isolation technique was adapted in
order to examine the question of DNA precursor
compartmentation in mammalian cells. By using this method
a reproducible proportion of the cellular nucleotides
remained associated with the isolated nuclei.
Examination, at several different cell densities, of
exponentially growing HeLa cells showed that...
Bacteriophage T4 gene 42 encodes dCMP hydroxymethylase, an
enzyme unique to the deoxyribonucleotide metabolism of T-even
bacteriophages. To study biochemical and biophysical properties of
the enzyme, as well as the interaction of dCMP hydroxymethylase with
other DNA precursor biosynthetic enzymes in vitro, availability of large
amounts of the enzyme is...
Imbalanced deoxyribonucleoside triphosphate (dNTP) pools are mutagenic for DNA synthesis in both intact cells and cell-free replication systems. Almost certainly, such mutagenesis involves competition between correctly and incorrectly base-paired precursors at replication sites. However, there are certain differences between the intact cell and cell-free systems that do not always allow...
Vaccinia virus-infected animal cells have been used to
study the interactions between the replication of
deoxyribonucleic acid (DNA) and the biosynthesis of its
nucleotide precursors. Some antimetabolites that inhibit
DNA replication have as their targets enzymes of nucleotide
biosynthesis. Furthermore, the disruption of nucleotide
metabolism can alter the fidelity of...
Although the synthesis of DNA precursors is
closely coordinated with DNA replication, it is still
not clear whether deoxyribonucleoside triphosphates
(dNTPs) influence DNA replication independently of
their interactions with DNA polymerase catalytic sites.
In an effort to understand the extent to which rate and
fidelity of DNA replication are regulated...