Section: Rotations

Michael Volkert, Ph.D.

Academic Role: Professor

Faculty Appointment(s) In:
   Molecular Genetics and Microbiology

Other Affiliation(s):
   Bacterial Genetics and Pathogenesis

Rotation Projects

Construction and testing of OXR1 inhibitory RNAs.  The OXR1 gene is induced by oxidative stress and localizes to mitochondria in human and yeast cells.  In yeast we have shown that OXR1 is required for resistance to treatments with the oxidative DNA damaging agent hydrogen peroxide and that resistance can be restored to the yeast mutant strain by expression of human OXR1.  This result suggests that OXR1 function is conserved from yeast to human forms and further suggests a role for human OXR1 in resistance to oxidative stress.  We will now test this directly by expressing short inhibitory RNAs (siRNA) in human cells and testing for oxidation sensitivity.  To accomplish this, we have identified and produced a set of siRNAs.  These siRNA expressing oligonucleotides now need to be cloned into the appropriate expression vectors and introduced into human HeLa cells.  The cells then need to be tested for OXR1 expression by western blotting methods to screen for inhibition using the anti OXR1 antibody.  If inhibition is detected, then the cells will be tested for resistance to hydrogen peroxide and other DNA oxidizing agents.

Genetic analysis of the human OXR2 gene. Higher eukaryotes have more than one OXR gene, whereas lower eukaryotes such as yeast have only one. The human OXR2 gene is a paralog of the human OXR1 gene discovered in our recent search for DNA protection genes. This gene is about 50% identical to human OXR1 and about 65% similar. OXR2 has been cloned. A rotation project will involve the comparison of this gene with OXR1 in terms of its ability to complement the mutator phenotype of a bacterial mutM mutY strain and to assess its ability to complement the oxidation sensitivity of the yeast OXR deletion mutant strain.

Analysis of dominant negative alleles of human PC4. Yeast cells lacking their PC4 ortholog Sub1 are sensitive to treatments with the oxidative agent hydrogen peroxide. Since human PC4 is able to protect bacterial cells from mutagenesis by oxidative agents and is able to complement the peroxide sensitivity of a yeast sub1 deletion mutant strain, PC4 function appears to be conserved from the yeast to the human forms.  This role for PC4 requires its DNA binding activity and our in vitro studies indicate that human PC4 functions in DNA repair.  Therefore human PC4 is likely to function in repair of oxidative DNA damage in mammalian cells.  Since PC4 appears to be a stable protein, it may be a poor candidate for siRNA inhibition methods.  Therefore we will test several potential dominant negative alleles of human PC4 for their ability to inhibit the function of the wild type allele.  PC4 works as a dimer to bind to unpaired regions of double-stranded DNA. We have several mutant alleles of PC4 that were produced by site-directed mutagenesis, that specifically inactivate PC4’s DNA binding activity. These mutant alleles should form heterodimers with the wild type PC4 protein and their over-expression should result in a predominance of dimers containing one mutant subunit. Since binding to DNA by dimers is generally cooperative, the binding affinity of a dimer is drastically reduced by the presence of a defective subunit.  Thus. Over-expression of a mutant PC4 allele should inactivate or severely impair the DNA binding required for its oxidation resistance. We will test the ability of the mutant alleles to inhibit PC4 function by co-expressing mutant and wild type alleles in yeast and testing for loss of complementation of peroxide resistance in the sub1 deletion mutant, and/or by co-expression in bacteria and testing for loss of the PC4 antimutator function seen in the mutM mutY strain of E. coli. If PC4 inactivation by the mutant PC4 alleles is seen in these tests, then the mutant alleles of PC4 will be expressed in HeLa cells and the transfected cells tested for loss of peroxide resistance.

Office: S6-117
Phone: 508-856-2314
E-mail: Michael.Volkert@umassmed.edu
Keywords: Human Cells, Yeast, DNA Damage and Repair, Mutagenesis, Genetics

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