Conspectus
DNA repair enzymes continuously
provide surveillance throughout
our cells, protecting the enclosed DNA from the damage that is constantly
arising from oxidation, alkylating species, and radiation. Members
of this enzyme class are intimately linked to pathways controlling
cancer and inflammation and are promising targets for diagnostics
and future therapies. Their study is benefiting widely from the development
of new tools and methods aimed at measuring their activities. Here,
we provide an Account of our laboratory’s work on developing
chemical tools to study DNA repair processes in vitro, as well as in cells and tissues, and what we have learned by applying
them.
We first outline early work probing how DNA repair enzymes
recognize
specific forms of damage by use of chemical analogs of the damage
with altered shapes and H-bonding abilities. One outcome of this was
the development of an unnatural DNA base that is incorporated selectively
by polymerase enzymes opposite sites of missing bases (abasic sites)
in DNA, a very common form of damage.
We then describe strategies
for design of fluorescent probes targeted
to base excision repair (BER) enzymes; these were built from small
synthetic DNAs incorporating fluorescent moieties to engender light-up
signals as the enzymatic reaction proceeds. Examples of targets for
these DNA probes include UDG, SMUG1, Fpg, OGG1, MutYH, ALKBH2, ALKBH3,
MTH1, and NTH1. Several such strategies were successful and were applied
both in vitro and in cellular settings; moreover,
some were used to discover small-molecule modulators of specific repair
enzymes. One of these is the compound SU0268, a potent OGG1 inhibitor
that is under investigation in animal models for inhibiting hyperinflammatory
responses.
To investigate cellular nucleotide sanitation pathways,
we designed
a series of “two-headed” nucleotides containing a damaged
DNA nucleotide at one end and ATP at the other; these were applied
to studying the three human sanitation enzymes MTH1, dUTPase, and
dITPase, some of which are therapeutic targets. The MTH1 probe (ARGO)
was used in collaboration with oncologists to measure the enzyme in
tumors as a disease marker and also to develop the first small-molecule
activators of the enzyme.
We proceed to discuss the development
of a “universal”
probe of base excision repair processes (UBER), which reacts covalently
with abasic site intermediates of base excision repair. UBER probes
light up in real time as the reaction occurs, enabling the observation
of base excision repair as it occurs in live cells and tissues. UBER
probes can also be used in efficient and simple methods for fluorescent
labeling of DNA. Finally, we suggest interesting directions for the
future of this field in biomedicine and human health.