Presenter Information

Savannah WhitfieldFollow

Presentation Type

Poster - Campus Access Only

Abstract

Endonuclease VIII (nei) is a DNA repair enzyme that cuts single damaged bases out of DNA. In Escherichia coli (E. coli), it is made along with four other genes (in the nei operon), but the functions of the other genes are unknown. Chromate is an environmental toxicant that creates the kind of DNA damage that is recognized by the repair protein encoded by the nei gene. In this study, we wanted to see if we could use chromate to investigate the function of the other four genes that are made at the same time.

To test this, single gene deletion mutants were treated with chromate and the effects on cell growth and DNA lesion (damage) formation were measured. Mutant E. coli missing the ybgL gene, the gene encoded immediately before nei, were found to be resistant to chromate. This suggests that the toxic DNA damage created by chromate needs ybgL to form in DNA. Double gene deletion mutants have also been generated to see if the effects are amplified when two genes are missing, indicating that the genes may be working together to repair or metabolize DNA damage.

More detailed in vitro studies on ybgL have been performed. The gene was isolated and put into an expression vector to produce the ybgL protein to study. Purified ybgL protein was incubated with different types of oxidized DNA damage to see its effects. Results indicated the ybgL protein has strong nuclease activity. This is the first indication of any function for this gene. We are now developing an in vitro assay using fluorescently labeled oligonucleotides. Studying the function of the nei operon and the ybgL protein to determine what they do for cells is a new discovery in the knowledge of DNA and protein interaction.

Category

Life Sciences

Available for download on Sunday, April 07, 2019

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Apr 15th, 3:00 PM Apr 15th, 4:00 PM

Studying Structure and Function of ybgL – an E. coli protein of unknown function

Endonuclease VIII (nei) is a DNA repair enzyme that cuts single damaged bases out of DNA. In Escherichia coli (E. coli), it is made along with four other genes (in the nei operon), but the functions of the other genes are unknown. Chromate is an environmental toxicant that creates the kind of DNA damage that is recognized by the repair protein encoded by the nei gene. In this study, we wanted to see if we could use chromate to investigate the function of the other four genes that are made at the same time.

To test this, single gene deletion mutants were treated with chromate and the effects on cell growth and DNA lesion (damage) formation were measured. Mutant E. coli missing the ybgL gene, the gene encoded immediately before nei, were found to be resistant to chromate. This suggests that the toxic DNA damage created by chromate needs ybgL to form in DNA. Double gene deletion mutants have also been generated to see if the effects are amplified when two genes are missing, indicating that the genes may be working together to repair or metabolize DNA damage.

More detailed in vitro studies on ybgL have been performed. The gene was isolated and put into an expression vector to produce the ybgL protein to study. Purified ybgL protein was incubated with different types of oxidized DNA damage to see its effects. Results indicated the ybgL protein has strong nuclease activity. This is the first indication of any function for this gene. We are now developing an in vitro assay using fluorescently labeled oligonucleotides. Studying the function of the nei operon and the ybgL protein to determine what they do for cells is a new discovery in the knowledge of DNA and protein interaction.