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Mutation of the genome is a problem which all organisms must contend with. As a result, there is a host of mismatch repair (MMR) mechanisms to aid in
detection of random mismatches which arise during replication. These errors, however, are not the only mutational events which organisms face. Oftentimes,
nucleotides or their precursors are damaged in a way which halts replication, as in the case of thymine dimers which result from UV damage. In this case, it
is up to translesion synthesis (TLS) to extend the replication machinery. However, much of the effects of nucleotide damage in vivo is unknown. One example
is damage due to reactive oxygen species in the cell. For instance, 8-oxo-2’-deoxyguanosine often leads to a G:C◊T:A transversion event. In our experiment,
we measure 8-oxodG’s rate of mispair in vivo as well as the frequency with which 8-oxodG is repaired when placed in an 8-oxodG/G as well as an 8-oxodG/C
mispair.
How oxidative damage works
Although cells are subject to many toxins, perhaps the most ubiquitous of these are reactive oxygen species. Created as by-products of many reactions,
including aerobic respiration, these agents are a potential danger to the genome due to the ease in which they react with a range of molecules, including DNA
nucleotides and their precursors. Take, for instance, a damaged guanosine, 8-oxo-dGTP, which frequently mispairs with both cytosine and adenine. Below is a
schematic for how this type of oxidative damage might alter the genetic code.
Assay Setup
Wildtype Sequence
Above is the wildtype sequence for TRP5, which we have moved to chromosome three, so as to be near a replication fork. In this region, there are several locations
which can contain silent mutations, if the bases highlighted above are changed.
Most importantly, the function of TRP5 in the strains above has been disrupted; the only way to restore TRP5 is to induce a C-C mismatch at the region (148)
indicated in red. Furthermore, the changes made in the two strains above created several degenerate codons; if mismatches occur in the third base-pairs of
any of these codons, all results should prove viable. Therefore, we can transform the above strains with oligonucleotides containing oxidatively damaged bases
in the degenerate positions and a cytosine which will line up in the 148th position.
SS-Oligo Transformation
Aside from random mutations, the only way for strain 2114 to regain function of TRP5 is to incorporate an oligonucleotide which creates a C-C mispair. If this
mispair is made, two results can occur; either the oligo is rejected and the original sequence of GAA is kept, or the oligo is kept and the original sequence is
GAA is changed to CAA. Only the latter will result in a TRP5+ colony. However, if the oligonucleotide is kept, a downstream 8-oxo-dG will also be incorporated,
opposite a degenerate base. The cell’s reaction to this mismatch can be studied by sequencing revertants.
The same C-C mispair is required for 2186 to regain TRP5 function. However, the 8-oxo-dG being studied is further downstream, and is opposite a
cytosine instead of a guanine.
Twenty TRP5+ rever-tants were sequenced after electroporation with a single- stranded oligonucleotide. For the most part, differences from the genomic
sequence did not occur, especially in the 2185 strain. This suggests that cellular mechanisms are proficient in detecting 8-oxo-dG/C mispairs.
However, repair mechanisms seemed less capable when detecting 8-oxo-dG/G mispairs in the 2114 strain, where it appears some mispair was not detected.
However, more sequences need to be run in order to determine whether 8-oxo-dG/G mispairs are really detected less efficiently.
The preliminary experiments demonstrated here have shown that oxidatively damaged oligonucleotides can be used to probe the function of cellular machinery.
One of the immediate next steps is to disrupt the function of Ogg1, a glycosylase which eliminates 8-oxo-dG bases opposite a C. By doing so, we knock out the
only known repair mechanism for 8-oxo-dG/C mispairs; therefore, we can determine whether 8-oxo-dG/C mispairs are in fact repaired efficiently to G/C pairs or
whether 8-oxo-dG/C mispairs simply 'slip through the cracks' and is a viable mispair which the cell allows. In the case of the latter, we should see little change in
reversions of the 2186 strain because repair was never a significant contributor. However, if the former is true and the cell is actively repairing 8-oxo-dG
mispairs, then we should see an increase in substitution of different bases due to an overload on repair mechanisms. If this is the case, mismatch repair
proteins such as MutSα and MutSβ can then be disrupted to clarify the role of such mismatch repair mechanisms in dealing with oxidatively damaged bases.
Special thanks to: Dr. Gray Crouse and David Doo
This poster was made possible by: Cathy Quinones, Pat Marsteller, the Howard Hughes Medical Institute under Grant No. 52005873, and supporters like you!
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