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Exploring Tripartite Interaction Between Yoaa, Holc, and SSB, Required for Repair of DNA Damage Made by AZT

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Exploring Tripartite Interaction Between Yoaa, Holc, and SSB, Required for Repair of DNA Damage Made by AZT Exploring tripartite interaction between YoaA, HolC, and SSB, required for repair of DNA damage made by AZT Una Milovanovic, Sneha Sathish, Margaret Hibnick, Mentor: Doctor Linda Bloom Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, FL Introduction The structure of DNA is constantly being challenged by chemicals that are naturally present in cells including oxygen and water. It has been estimated that 20,000 DNA damaging events occur within a cell in a 24 hour period. If those damages do not get fixed, the genetic code will be altered and chromosomes may break. This issue is addressed by cell mechanisms where the damaged bit of DNA is removed and the undamaged DNA strand is used as a template to remake the damaged strand. However, there is some damage that does not get repaired prior to DNA replication. In this case, cell have to conduct special mechanisms to either fix the damaged DNA during replication or bypass the damage and fix it later. Our collaborators in the Lovett laboratory at Brandeis University use genetic approaches in Escherichia coli, a model organism, to discover processes that can repair DNA damage during replication. They recently discovered a pathway that requires at least two proteins, HolC and YoaA, to give cells tolerance to DNA damage created by 3’-azido- 3’-thymidine (AZT). AZT is a chain terminator so that when it is incorporated into DNA by a DNA polymerase, the DNA polymer cannot be extended any further. Thus, DNA replication is blocked. When the polymerase stops, single-stranded DNA starts to accumulate and is bound by a protein called single-stranded DNA binding protein or SSB. The Lovett laboratory discovered a new gene, YoaA, that gives cells tolerance to AZT. Prior to this study, the function of the YoaA gene was not known. Based on its sequence, YoaA encodes a DNA helicase, and enzyme that unwinds DNA strands. Hypothesis or Approach Based on the sequence of the YoaA gene, we expect the YoaA protein to be DNA helicase that is related to another DNA helicase in E.coli named DinG, with its role to unwind the damaged DNA so that a nuclease can cleave the AZT from the end. Furthermore, we hypothesize that there is a tripartite interaction, YoaA-HolC-SSB, that is needed for YoaA to bump the polymerase off DNA and unwind DNA. Methodology/Future Work Initially we will make the DNA substrates for this project and purify DinG and YoaA. After we have all these reagents in hand, we will perform helicase assays to determine how HolC, SSB and the combination of HolC and SSB affect YoaA helicase activity by mixing YoaA with different combinations of HolC and SSB to determine whether HolC and/or SSB affect the ability of YoaA to unwind DNA . We also have HolC and SSB mutant proteins that cannot interact with each other. If a HolC-SSB complex is needed for YoaA to unwind DNA, the mutants which cannot make this complex should not help YoaA unwind DNA. The assay that we will use is a fluorescence assay in which a fluorescein molecule is attached to each strand of duplex DNA. When the DNA is in the double-stranded form, the fluorescein molecules and interact and the fluorescence will be quenched (low fluorescence signal). When YoaA unwinds DNA and the DNA becomes single-stranded, the fluorescein molecules will be separated so the fluorescence will increase. DinG is known to interact with SSB, and SSB stimulates the helicase activity of DinG. In these studies, we will use DinG for two purposes. First, DinG will be used for technical purposes as a positive control to show that our assays are working. We know DinG unwinds DNA and we know that SSB makes DinG unwind DNA better, so if our experiments are working correctly this is what we should see. Second, DinG will answer a scientific question. DinG is related to YoaA, but DinG does not protect cells from AZT as well as YoaA does. Therefore, we want to see if there are any differences in activity that could explain this. We will measure the DNA helicase activity of YoaA and compare it to DinG helicase activity. By doing so, we will be working to define the substrate specificity of YoaA. By testing the helicase activity of YoaA on this type of DNA substrate as well as other substrates on which XPD-family helicases act including forked DNA substrates and D-loops. If we see that DinG unwinds a DNA substrate, but YoaA does not, then we can conclude that our assay is working and that YoaA is not active in unwinding that particular DNA substrate. DNA helicases are specialized, meaning that they have different preferences for the types of DNA structures that they unwind. This suggest that DinG and YoaA may have some differences in the DNA structures that they prefer to unwind. By doing this, we will be able to compare the activities of YoaA and DinG on several DNA structures to determine what the similarities and differences in their DNA specificities are. Results • We purified, labeled, and concentrated DNA. • As we proceed to conduct our experiment, we will generate more results later in the future. Applications • To make clinical correlations between malfunctions in these biochemical pathways and disease. – e.g. colorectal cancer (HNPCC) and mismatch repair. • To develop tools for biomedical research and diagnostics – PCR – DNA sequencing – Site-directed mutagenesis – Cloning techniques. • To develop therapeutics based on inhibiting DNA replication/metabolism. References -- Lovett laboratory at Brandeis University -- Professor Linda Bloom Department of Biochemistry and Molecular Biology at the University of Florida .
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