Background Abstract Results Conclusion Methods
Undergraduate Category: Physical and Life Sciences Degree Level: Bachelor's Abstract ID# 1183
Directed Evolution of DNA Pol IV to Acquire New Functions Abstract
Jennifer Taylor, Caitlin Kramer, Penny J Beuning
Background
Goal
Mutate DinB in order to help it bypass the dimer DNA adducts that UmuC is able to bypass naturally.
Results
Mutated DinB Plasmid Survival Compared to Original DinB Plasmid
Methods
1.00E+00
Survival of Mutated Plasmids vs. Original Plasmids
1.00E-01 Survival
1.00E+00
UmuC
1.00E-01
Mutated DinB
1.00E-02 1.00E-03 1.00E-04
Original DinB
1.00E-05
HA Mutagenesis
1.00E-06 0
50 75 Dose (J/m2)
100
Mutated DinB-CC Plasmid Survival Compared to Original DinB-CC Plasmid
1.00E-02
1.00E+00
Survival
1.00E-01
1.00E-03
1.00E-02 1.00E-03
Mutated DinB-CC
1.00E-04
Original DinB-CC
1.00E-05 1.00E-06
0
50
75
100
Dose (J/m2) 1.00E-04
Mutated DinB-LFCC Plasmid Survival Compared to OriginalDinB-LFCC Plasmid 1.00E+00
Mutated DinB
1.00E-05
1.00E-01 Mutated DinB-CC UmuC empty vector Original DinB Original DinB-LFCC Original DinB-CC
1.00E-06 0
50
75
Dose J/m2
- SOS response is a mechanism that activates over 40 genes allowing for survival in a hostile environment - DNA polymerase Y-family able to bypass lesions in the DNA template - DNA polymerase IV (DinB) and V (UmuC) are able to perform translesion DNA synthesis in which correct or incorrect dNMPs are inserted in the opposite template lesions.
Mutated DinB-LFCC DinB empty vector
100
Survival
Survival
All organisms are exposed to both endogenous and exogenous stresses that can lead to DNA damage. The E. coli SOS response is a DNA damage response mechanism that results in the expression of proteins involved in DNA recombination, repair, and translesion DNA synthesis. Y-family DNA polymerases are able to bypass lesions in the DNA template due to their relatively open active sites. Two Y-family members in E. coli include DNA polymerase IV (DinB) and DNA polymerase V (UmuD’2C), which are specialized to bypass different types of DNA lesions. DNA polymerase V is capable of bypassing thymine-thymine dimers caused by UV light, while DNA polymerase IV is able to bypass adducts that form in the minor groove of DNA. DinB and UmuC have different structural domains which could account for their differences in bypassing distinct lesions. Their little finger domains are structurally different, for example, UmuC also has an additional C-terminal domain (CTD), which could contribute to its ability to bypass certain lesions. Cells lacking Pol V show increased sensitivity to UV exposure due to their inability to bypass thyminethymine dimers. Directed evolution of DinB and DinB-UmuC chimeras that result in enhanced survival to UV exposure would elucidate polymerase structural requirements for UV lesion bypass. Hydroxylamine mutagenesis is used to generate libraries of mutagenized DinB constructs, which are then assayed for increased UV tolerance relative to wild-type DinB. Constructs that show improved performance will be subjected to further rounds of mutagenesis and UV exposure to further hone functional changes.
1.00E-02 1.00E-03
- Hydroxylamine modifies base structures leading to mispairing. - Ethanol precipitation method for hydroxylamine in vitro mutagenesis.
UV Exposure - 0, 50, 75 and 100 J/m2 exposure per plasmid. - After indicated exposure, plate serial dilutions. - Killing Curve to analyze survival.
DinB-LFCC HA
1.00E-04
DinB-LFCC
1.00E-05 1.00E-06 0
50
75
100
Dose (J/m2)
Conclusion
The results obtained show that the mutated wild type DinB along with DinB-CC, and DinB-LFCC is yet to improve the survival rate when compared to the original non-mutated plasmids. DinB-LFCC shows the more promising increasing survival. The ongoing research project will continue to explore if the mutated plasmids can show significant survival to UV exposure and if DinB is successfully mutated then it would be capable of bypassing the dimer DNA adducts that UmuC is able to bypass naturally.
References Hawver, Lisa A., Mohammad Tehrani, Nicole M. Antczak, Danielle Kania, Stephanie Muser, Jana Sefcikova, and Penny J. Beuning. "Point Mutations in Escherichia Coli DNA Pol V That Confer Resistance to Non-cognate DNA Damage Also Alter Protein-protein Interactions." Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis (2015) 1-14.
Acknowledgments
Directed Evolution of DNA Pol IV to Acquire New Functions Abstract
Jennifer Taylor, Caitlin Kramer, Penny J Beuning
Background
Goal
Mutate DinB in order to help it bypass the dimer DNA adducts that UmuC is able to bypass naturally.
Results
Mutated DinB Plasmid Survival Compared to Original DinB Plasmid
Methods
1.00E+00
Survival of Mutated Plasmids vs. Original Plasmids
1.00E-01 Survival
1.00E+00
UmuC
1.00E-01
Mutated DinB
1.00E-02 1.00E-03 1.00E-04
Original DinB
1.00E-05
HA Mutagenesis
1.00E-06 0
50 75 Dose (J/m2)
100
Mutated DinB-CC Plasmid Survival Compared to Original DinB-CC Plasmid
1.00E-02
1.00E+00
Survival
1.00E-01
1.00E-03
1.00E-02 1.00E-03
Mutated DinB-CC
1.00E-04
Original DinB-CC
1.00E-05 1.00E-06
0
50
75
100
Dose (J/m2) 1.00E-04
Mutated DinB-LFCC Plasmid Survival Compared to OriginalDinB-LFCC Plasmid 1.00E+00
Mutated DinB
1.00E-05
1.00E-01 Mutated DinB-CC UmuC empty vector Original DinB Original DinB-LFCC Original DinB-CC
1.00E-06 0
50
75
Dose J/m2
- SOS response is a mechanism that activates over 40 genes allowing for survival in a hostile environment - DNA polymerase Y-family able to bypass lesions in the DNA template - DNA polymerase IV (DinB) and V (UmuC) are able to perform translesion DNA synthesis in which correct or incorrect dNMPs are inserted in the opposite template lesions.
Mutated DinB-LFCC DinB empty vector
100
Survival
Survival
All organisms are exposed to both endogenous and exogenous stresses that can lead to DNA damage. The E. coli SOS response is a DNA damage response mechanism that results in the expression of proteins involved in DNA recombination, repair, and translesion DNA synthesis. Y-family DNA polymerases are able to bypass lesions in the DNA template due to their relatively open active sites. Two Y-family members in E. coli include DNA polymerase IV (DinB) and DNA polymerase V (UmuD’2C), which are specialized to bypass different types of DNA lesions. DNA polymerase V is capable of bypassing thymine-thymine dimers caused by UV light, while DNA polymerase IV is able to bypass adducts that form in the minor groove of DNA. DinB and UmuC have different structural domains which could account for their differences in bypassing distinct lesions. Their little finger domains are structurally different, for example, UmuC also has an additional C-terminal domain (CTD), which could contribute to its ability to bypass certain lesions. Cells lacking Pol V show increased sensitivity to UV exposure due to their inability to bypass thyminethymine dimers. Directed evolution of DinB and DinB-UmuC chimeras that result in enhanced survival to UV exposure would elucidate polymerase structural requirements for UV lesion bypass. Hydroxylamine mutagenesis is used to generate libraries of mutagenized DinB constructs, which are then assayed for increased UV tolerance relative to wild-type DinB. Constructs that show improved performance will be subjected to further rounds of mutagenesis and UV exposure to further hone functional changes.
1.00E-02 1.00E-03
- Hydroxylamine modifies base structures leading to mispairing. - Ethanol precipitation method for hydroxylamine in vitro mutagenesis.
UV Exposure - 0, 50, 75 and 100 J/m2 exposure per plasmid. - After indicated exposure, plate serial dilutions. - Killing Curve to analyze survival.
DinB-LFCC HA
1.00E-04
DinB-LFCC
1.00E-05 1.00E-06 0
50
75
100
Dose (J/m2)
Conclusion
The results obtained show that the mutated wild type DinB along with DinB-CC, and DinB-LFCC is yet to improve the survival rate when compared to the original non-mutated plasmids. DinB-LFCC shows the more promising increasing survival. The ongoing research project will continue to explore if the mutated plasmids can show significant survival to UV exposure and if DinB is successfully mutated then it would be capable of bypassing the dimer DNA adducts that UmuC is able to bypass naturally.
References Hawver, Lisa A., Mohammad Tehrani, Nicole M. Antczak, Danielle Kania, Stephanie Muser, Jana Sefcikova, and Penny J. Beuning. "Point Mutations in Escherichia Coli DNA Pol V That Confer Resistance to Non-cognate DNA Damage Also Alter Protein-protein Interactions." Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis (2015) 1-14.
Acknowledgments
