Abstract

TranslesionDNAsynthesis(TLS)polymerasesarecriticaltocellsurvivalbyreplacinghighfidelitypolymerases duringroadblocksthatoccurduringDNArepairandreplication.Onedifficultyinelucidatingthemultiplerolesof thesepolymerasesisthatitisimpossibletoidentifywhichpolymeraseisactiveinaspecificsituation.Herewe proposeachemicalbiologyapproachinwhichwecanmeasuretheactivityofDNApolymerasekappa.We havedesignedandsynthesizedN2-benzyl-2?-deoxyguanosineandanalogsthatarehighlyselecttowardpol kappa.Wehavepreviouslyshownthatinvitro,N2-benzyl-GTPreactswithpol?105-foldmoreefficientlythan poleta,iota,beta,nu,anddelta,andincells,theincorporationofN2-4-ethynylbenzyl-dGintotheDNAis dependentonpolkappa.Withthistoolwewillexaminethemultiplerolesofpolkappaintwofollowingspecific aims:(1)DeterminetheroleofpolkappainNER,and(2)determinetherolepolkappaplaysduringS-phase.
In aim1, wewillexaminetheNERactivityofpolkappawithrespecttoDNAdamage,protein-protein interactions,andthelocationoftheactivityinthegenome.
In aim2 wewillexaminepolkappaactivityinS- phasewithrespecttobypassingDNAdamageandreplicationofnon-BDNAsequences.Inparticularwewill examinethepolymeraseswitchmechanismsatthereplicationfork,theroleofprotein-proteininteractionsin activationofpolkappaactivity,andthelocationofpolkappaactivityinthegenome.Similartechniqueswillbe employedinthetwoaims.(i)ActivityassayswillbeperformedutilizingN2-4-ethynylbenzyl-dGandClick Chemistrytoattachafluorophore.Theactivitywillbeanalyzedbyfluorescencemicroscopytoexamine nuclear/cytoplasmiclocalizationof4-ethynylbenzyl-dG,whileflowcytometrywillbeusedtoexaminecell-cycle activity.(ii)Thesetwotechniqueswillbecombinedwithmutant-inactive-proteinstodeterminethecritical proteinsandinteractionsinvolvedintheactivity.(iii)iPOND-likeexperimentswillbeperformedtoidentify proteinsassociatedwiththeactivityinanunbiasedmanner.(iv)DNAstrandfiberassayswillbeemployedto distinguishbetweenthepolymeraseswitchmechanismandpost-gaprepairduringS-phase.(v)Next generationsequencingwillbeutilizedtoprobethegenomicidentityoftheactivity.Thisproposalisvery innovativeincreatinganewmethodbywhichscientistswillbeabletoexaminetheactivityofasingleDNA polymeraseinacell. PUBLICHEALTH

Public Health Relevance

.DifferencesinactivityofDNApolymerasehaveamajorimpactontheability ofanindividualtorespondtoDNAdamagingagents.Thismethodologymaybeusedinidentifyingthe susceptibilityofindividualororganstocarcinogensandtheefficacyofDNAdamagingchemotherapeutic agents.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
3R01ES021762-08S1
Application #
9944019
Study Section
Cancer Etiology Study Section (CE)
Program Officer
Tyson, Frederick L
Project Start
2012-07-20
Project End
2023-05-31
Budget Start
2020-07-01
Budget End
2021-05-31
Support Year
8
Fiscal Year
2020
Total Cost
Indirect Cost

  Institution

Name
Pennsylvania State University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
129348186
City
Hershey
State
PA
Country
United States
Zip Code
17033

  Publications

Prakasha Gowda, A S; Spratt, Thomas E (2017) Active Site Interactions Impact Phosphoryl Transfer during Replication of Damaged and Undamaged DNA by Escherichia coli DNA Polymerase I. Chem Res Toxicol 30:2033-2043
Basu, Ashis K; Pande, Paritosh; Bose, Arindam (2017) Translesion Synthesis of 2'-Deoxyguanosine Lesions by Eukaryotic DNA Polymerases. Chem Res Toxicol 30:61-72
Gowda, A S Prakasha; Krzeminski, Jacek; Amin, Shantu et al. (2017) Mutagenic Replication of N2-Deoxyguanosine Benzo[a]pyrene Adducts by Escherichia coli DNA Polymerase I and Sulfolobus solfataricus DNA Polymerase IV. Chem Res Toxicol 30:1168-1176
Gowda, A S Prakasha; Suo, Zucai; Spratt, Thomas E (2017) Honokiol Inhibits DNA Polymerases ? and ? and Increases Bleomycin Sensitivity of Human Cancer Cells. Chem Res Toxicol 30:715-725
Gowda, A S Prakasha; Lee, Marietta; Spratt, Thomas E (2017) N2 -Substituted 2'-Deoxyguanosine Triphosphate Derivatives as Selective Substrates for Human DNA Polymerase ?. Angew Chem Int Ed Engl 56:2628-2631
Patra, Amritraj; Politica, Dustin A; Chatterjee, Arindom et al. (2016) Mechanism of Error-Free Bypass of the Environmental Carcinogen N-(2'-Deoxyguanosin-8-yl)-3-aminobenzanthrone Adduct by Human DNA Polymerase??. Chembiochem 17:2033-2037
Gowda, A S Prakasha; Spratt, Thomas E (2016) DNA Polymerase ? Rapidly Bypasses O6-Methyl-dG but Not O6-[4-(3-Pyridyl)-4-oxobutyl-dG and O2-Alkyl-dTs. Chem Res Toxicol 29:1894-1900
Gowda, A S Prakasha; Spratt, Thomas E (2016) DNA Polymerases ? and ? Combine to Bypass O(2)-[4-(3-Pyridyl)-4-oxobutyl]thymine, a DNA Adduct Formed from Tobacco Carcinogens. Chem Res Toxicol 29:303-16
Gowda, A S Prakasha; Moldovan, George-Lucian; Spratt, Thomas E (2015) Human DNA Polymerase ? Catalyzes Correct and Incorrect DNA Synthesis with High Catalytic Efficiency. J Biol Chem 290:16292-303
Pande, Paritosh; Haraguchi, Kazuhiro; Jiang, Yu-Lin et al. (2015) Unlike catalyzing error-free bypass of 8-oxodGuo, DNA polymerase ? is responsible for a significant part of Fapy·dG-induced G ? T mutations in human cells. Biochemistry 54:1859-62

Showing the most recent 10 out of 17 publications