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Article ; Online: Inhibition of tryptophan 2,3-dioxygenase impairs DNA damage tolerance and repair in glioma cells.

Reed, Megan R / Maddukuri, Leena / Ketkar, Amit / Byrum, Stephanie D / Zafar, Maroof K / Bostian, April C L / Tackett, Alan J / Eoff, Robert L

NAR cancer

2021 Volume 3, Issue 2, Page(s) zcab014

Abstract: Expression of tryptophan 2,3-dioxygenase (TDO) is a determinant of malignancy in gliomas through kynurenine (KYN) signaling. We report that inhibition of TDO activity attenuated recovery from replication stress and increased the genotoxic effects of bis- ... ...

Abstract	Expression of tryptophan 2,3-dioxygenase (TDO) is a determinant of malignancy in gliomas through kynurenine (KYN) signaling. We report that inhibition of TDO activity attenuated recovery from replication stress and increased the genotoxic effects of bis-chloroethylnitrosourea (BCNU). Activation of the Chk1 arm of the replication stress response (RSR) was reduced when TDO activity was blocked prior to BCNU treatment, whereas phosphorylation of serine 33 (pS33) on replication protein A (RPA) was enhanced-indicative of increased fork collapse. Analysis of quantitative proteomic results revealed that TDO inhibition reduced nuclear 53BP1 and sirtuin levels. We confirmed that cells lacking TDO activity exhibited elevated gamma-H2AX signal and defective recruitment of 53BP1 to chromatin following BCNU treatment, which corresponded with delayed repair of DNA breaks. Addition of exogenous KYN increased the rate of break repair. TDO inhibition diminished SIRT7 deacetylase recruitment to chromatin, which increased histone H3K18 acetylation-a key mark involved in preventing 53BP1 recruitment to sites of DNA damage. TDO inhibition also sensitized cells to ionizing radiation (IR)-induced damage, but this effect did not involve altered 53BP1 recruitment. These experiments support a model where TDO-mediated KYN signaling helps fuel a robust response to replication stress and DNA damage.
Language	English
Publishing date	2021-04-09
Publishing country	England
Document type	Journal Article
ISSN	2632-8674
ISSN (online)	2632-8674
DOI	10.1093/narcan/zcab014
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Article ; Online: Human Rev1 relies on insert-2 to promote selective binding and accurate replication of stabilized G-quadruplex motifs.

Ketkar, Amit / Smith, Lane / Johnson, Callie / Richey, Alyssa / Berry, Makayla / Hartman, Jessica H / Maddukuri, Leena / Reed, Megan R / Gunderson, Julie E C / Leung, Justin W C / Eoff, Robert L

Nucleic acids research

2021 Volume 49, Issue 4, Page(s) 2065–2084

Abstract: We previously reported that human Rev1 (hRev1) bound to a parallel-stranded G-quadruplex (G4) from the c-MYC promoter with high affinity. We have extended those results to include other G4 motifs, finding that hRev1 exhibited stronger affinity for ... ...

Abstract	We previously reported that human Rev1 (hRev1) bound to a parallel-stranded G-quadruplex (G4) from the c-MYC promoter with high affinity. We have extended those results to include other G4 motifs, finding that hRev1 exhibited stronger affinity for parallel-stranded G4 than either anti-parallel or hybrid folds. Amino acids in the αE helix of insert-2 were identified as being important for G4 binding. Mutating E466 and Y470 to alanine selectively perturbed G4 binding affinity. The E466K mutant restored wild-type G4 binding properties. Using a forward mutagenesis assay, we discovered that loss of hRev1 increased G4 mutation frequency >200-fold compared to the control sequence. Base substitutions and deletions occurred around and within the G4 motif. Pyridostatin (PDS) exacerbated this effect, as the mutation frequency increased >700-fold over control and deletions upstream of the G4 site more than doubled. Mutagenic replication of G4 DNA (±PDS) was partially rescued by wild-type and E466K hRev1. The E466A or Y470A mutants failed to suppress the PDS-induced increase in G4 mutation frequency. These findings have implications for the role of insert-2, a motif conserved in vertebrates but not yeast or plants, in Rev1-mediated suppression of mutagenesis during G4 replication.
MeSH term(s)	Cell Line ; DNA/chemistry ; DNA/metabolism ; DNA Replication ; DNA-Directed DNA Polymerase/metabolism ; G-Quadruplexes ; Genes, myc ; Humans ; Models, Molecular ; Mutation ; Nucleotide Motifs ; Nucleotidyltransferases/chemistry ; Nucleotidyltransferases/genetics ; Nucleotidyltransferases/metabolism ; Protein Binding
Chemical Substances	DNA (9007-49-2) ; Nucleotidyltransferases (EC 2.7.7.-) ; REV1 protein, human (EC 2.7.7.-) ; DNA-Directed DNA Polymerase (EC 2.7.7.7)
Language	English
Publishing date	2021-02-09
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	186809-3
ISSN	1362-4962 ; 1362-4954 ; 0301-5610 ; 0305-1048
ISSN (online)	1362-4962 ; 1362-4954
ISSN	0301-5610 ; 0305-1048
DOI	10.1093/nar/gkab041
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Article ; Online: Inhibition of Human DNA Polymerases Eta and Kappa by Indole-Derived Molecules Occurs through Distinct Mechanisms.

Ketkar, Amit / Maddukuri, Leena / Penthala, Narsimha R / Reed, Megan R / Zafar, Maroof K / Crooks, Peter A / Eoff, Robert L

ACS chemical biology

2019 Volume 14, Issue 6, Page(s) 1337–1351

Abstract: Overexpression of human DNA polymerase kappa (hpol κ) in glioblastoma is associated with shorter survival time and resistance to the alkylating agent temozolomide (TMZ), making it an attractive target for the development of small-molecule inhibitors. We ... ...

Abstract	Overexpression of human DNA polymerase kappa (hpol κ) in glioblastoma is associated with shorter survival time and resistance to the alkylating agent temozolomide (TMZ), making it an attractive target for the development of small-molecule inhibitors. We previously reported on the development and characterization of indole barbituric acid-derived (IBA) inhibitors of translesion DNA synthesis polymerases (TLS pols). We have now identified a potent and selective inhibitor of hpol κ based on the indole-aminoguanidine (IAG) chemical scaffold. The most promising IAG analogue, IAG-10, exhibited greater inhibitory action against hpol κ than any other human Y-family member, as well as pols from the A-, B-, and X-families. Inhibition of hpol κ by IAG analogues appears to proceed through a mechanism that is distinct from inhibition of hpol η based on changes in DNA binding affinity and nucleotide insertion kinetics. By way of comparison, both IAG and IBA analogues inhibited binary complex formation by hpol κ and ternary complex formation by hpol η. Decreasing the concentration of enzyme and DNA in the reaction mixture lowered the IC
MeSH term(s)	Alkylation ; DNA Damage ; DNA-Directed DNA Polymerase/drug effects ; Enzyme Inhibitors/pharmacology ; Humans ; Indoles/pharmacology ; Inhibitory Concentration 50
Chemical Substances	Enzyme Inhibitors ; Indoles ; DNA-Directed DNA Polymerase (EC 2.7.7.7) ; POLK protein, human (EC 2.7.7.7) ; Rad30 protein (EC 2.7.7.7)
Language	English
Publishing date	2019-05-22
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, P.H.S.
ISSN	1554-8937
ISSN (online)	1554-8937
DOI	10.1021/acschembio.9b00304
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Article ; Online: A Small-Molecule Inhibitor of Human DNA Polymerase η Potentiates the Effects of Cisplatin in Tumor Cells.

Zafar, Maroof K / Maddukuri, Leena / Ketkar, Amit / Penthala, Narsimha R / Reed, Megan R / Eddy, Sarah / Crooks, Peter A / Eoff, Robert L

Biochemistry

2018 Volume 57, Issue 7, Page(s) 1262–1273

Abstract: Translesion DNA synthesis (TLS) performed by human DNA polymerase eta (hpol η) allows tolerance of damage from cis-diamminedichloroplatinum(II) (CDDP or cisplatin). We have developed hpol η inhibitors derived from N-aryl-substituted indole barbituric ... ...

Abstract	Translesion DNA synthesis (TLS) performed by human DNA polymerase eta (hpol η) allows tolerance of damage from cis-diamminedichloroplatinum(II) (CDDP or cisplatin). We have developed hpol η inhibitors derived from N-aryl-substituted indole barbituric acid (IBA), indole thiobarbituric acid (ITBA), and indole quinuclidine scaffolds and identified 5-((5-chloro-1-(naphthalen-2-ylmethyl)-1H-indol-3-yl)methylene)-2-thioxodihydropyrimidine-4,6(1H,5H)-dione (PNR-7-02), an ITBA derivative that inhibited hpol η activity with an IC
MeSH term(s)	Antineoplastic Agents/pharmacology ; Cell Line, Tumor ; Cisplatin/pharmacology ; DNA-Directed DNA Polymerase/metabolism ; Enzyme Inhibitors/chemistry ; Enzyme Inhibitors/pharmacology ; Humans ; Indoles/chemistry ; Indoles/pharmacology ; Molecular Docking Simulation ; Neoplasms/drug therapy ; Neoplasms/metabolism ; Pyrimidines/chemistry ; Pyrimidines/pharmacology ; Small Molecule Libraries/chemistry ; Small Molecule Libraries/pharmacology ; Thiobarbiturates/chemistry ; Thiobarbiturates/pharmacology
Chemical Substances	Antineoplastic Agents ; Enzyme Inhibitors ; Indoles ; Pyrimidines ; Small Molecule Libraries ; Thiobarbiturates ; DNA-Directed DNA Polymerase (EC 2.7.7.7) ; Rad30 protein (EC 2.7.7.7) ; thiobarbituric acid (M1YZW5SS7C) ; Cisplatin (Q20Q21Q62J)
Language	English
Publishing date	2018-01-30
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	1108-3
ISSN	1520-4995 ; 0006-2960
ISSN (online)	1520-4995
ISSN	0006-2960
DOI	10.1021/acs.biochem.7b01176
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Article ; Online: Human Translesion Polymerase κ Exhibits Enhanced Activity and Reduced Fidelity Two Nucleotides from G-Quadruplex DNA.

Eddy, Sarah / Tillman, Magdalena / Maddukuri, Leena / Ketkar, Amit / Zafar, Maroof K / Eoff, Robert L

Biochemistry

2016 Volume 55, Issue 37, Page(s) 5218–5229

Abstract: We have investigated the in vitro properties of human Y-family polymerase κ (hpol κ) on G-quadruplex DNA (G4 DNA). Similar to hpol η, another Y-family member implicated in replication of G4 motifs, hpol κ bound G4 DNA with a 5.7-fold preference over ... ...

Abstract	We have investigated the in vitro properties of human Y-family polymerase κ (hpol κ) on G-quadruplex DNA (G4 DNA). Similar to hpol η, another Y-family member implicated in replication of G4 motifs, hpol κ bound G4 DNA with a 5.7-fold preference over control, non-G4 DNA. Results from pol extension assays are consistent with the notion that G-quadruplexes present a stronger barrier to DNA synthesis by hpol κ than they do to that by hpol η. However, kinetic analysis revealed that hpol κ activity increases considerably when the enzyme is 2-3 nucleotides from the G4 motif, a trend that was reported previously for hpol η, though the increase was less pronounced. The increase in hpol κ activity on G4 DNA was readily observed in the presence of either potassium or sodium but much less so when lithium was used in the buffer. The increased activity 2-3 nucleotides from the G4 motif was accompanied by a decrease in the fidelity of hpol κ when the counterion was either potassium or sodium but not in the presence of lithium. The activity of hpol κ decreased progressively as the primer was moved closer than 2 nucleotides from the G4 motif when either potassium or sodium was used to stabilize the G-quadruplex. Interestingly, the decrease in catalytic activity at the site of the quadruplex observed in potassium-containing buffer was accompanied by an increase in fidelity on G4 substrates versus control non-G4 substrates. This trend of increased fidelity in copying a tetrad-associated guanine was observed previously for hpol η, but not for the B-family member hpol ε, which exhibited a large decrease in both efficiency and fidelity in the attempt to copy the first guanine in the G4 motif. In summary, hpol κ activity was enhanced relative to those of other Y-family members when the enzyme is 2-3 nucleotides from the G4 motif, but hpol κ appears to be less competent than hpol η at copying tetrad-associated guanines.
MeSH term(s)	DNA/metabolism ; DNA Damage ; DNA-Directed DNA Polymerase/metabolism ; Fluorescence Polarization ; G-Quadruplexes ; Humans ; Kinetics
Chemical Substances	DNA (9007-49-2) ; DNA-Directed DNA Polymerase (EC 2.7.7.7) ; POLK protein, human (EC 2.7.7.7)
Language	English
Publishing date	2016-09-20
Publishing country	United States
Document type	Journal Article
ZDB-ID	1108-3
ISSN	1520-4995 ; 0006-2960
ISSN (online)	1520-4995
ISSN	0006-2960
DOI	10.1021/acs.biochem.6b00374
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Article ; Online: The Werner syndrome protein limits the error-prone 8-oxo-dG lesion bypass activity of human DNA polymerase kappa.

Maddukuri, Leena / Ketkar, Amit / Eddy, Sarah / Zafar, Maroof K / Eoff, Robert L

Nucleic acids research

2014 Volume 42, Issue 19, Page(s) 12027–12040

Abstract: Human DNA polymerase kappa (hpol κ) is the only Y-family member to preferentially insert dAMP opposite 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG) during translesion DNA synthesis. We have studied the mechanism of action by which hpol κ activity is ... ...

Abstract	Human DNA polymerase kappa (hpol κ) is the only Y-family member to preferentially insert dAMP opposite 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG) during translesion DNA synthesis. We have studied the mechanism of action by which hpol κ activity is modulated by the Werner syndrome protein (WRN), a RecQ helicase known to influence repair of 8-oxo-dG. Here we show that WRN stimulates the 8-oxo-dG bypass activity of hpol κ in vitro by enhancing the correct base insertion opposite the lesion, as well as extension from dC:8-oxo-dG base pairs. Steady-state kinetic analysis reveals that WRN improves hpol κ-catalyzed dCMP insertion opposite 8-oxo-dG ∼10-fold and extension from dC:8-oxo-dG by 2.4-fold. Stimulation is primarily due to an increase in the rate constant for polymerization (kpol), as assessed by pre-steady-state kinetics, and it requires the RecQ C-terminal (RQC) domain. In support of the functional data, recombinant WRN and hpol κ were found to physically interact through the exo and RQC domains of WRN, and co-localization of WRN and hpol κ was observed in human cells treated with hydrogen peroxide. Thus, WRN limits the error-prone bypass of 8-oxo-dG by hpol κ, which could influence the sensitivity to oxidative damage that has previously been observed for Werner's syndrome cells.
MeSH term(s)	8-Hydroxy-2'-Deoxyguanosine ; DNA/biosynthesis ; DNA/metabolism ; DNA Adducts/metabolism ; DNA-Directed DNA Polymerase/metabolism ; Deoxyguanosine/analogs & derivatives ; Deoxyguanosine/metabolism ; Exodeoxyribonucleases/chemistry ; Exodeoxyribonucleases/metabolism ; HeLa Cells ; Humans ; Kinetics ; Protein Structure, Tertiary ; RecQ Helicases/chemistry ; RecQ Helicases/metabolism
Chemical Substances	DNA Adducts ; 8-Hydroxy-2'-Deoxyguanosine (88847-89-6) ; DNA (9007-49-2) ; DNA-Directed DNA Polymerase (EC 2.7.7.7) ; POLK protein, human (EC 2.7.7.7) ; Exodeoxyribonucleases (EC 3.1.-) ; RecQ Helicases (EC 3.6.4.12) ; Deoxyguanosine (G9481N71RO)
Language	English
Publishing date	2014-10-07
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	186809-3
ISSN	1362-4962 ; 1362-4954 ; 0301-5610 ; 0305-1048
ISSN (online)	1362-4962 ; 1362-4954
ISSN	0301-5610 ; 0305-1048
DOI	10.1093/nar/gku913
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Article ; Online: A Functional Precision Medicine Pipeline Combines Comparative Transcriptomics and Tumor Organoid Modeling to Identify Bespoke Treatment Strategies for Glioblastoma.

Cells

2021 Volume 10, Issue 12

Abstract: Li Fraumeni syndrome (LFS) is a hereditary cancer predisposition syndrome caused by germline mutations in TP53. TP53 is the most common mutated gene in human cancer, occurring in 30-50% of glioblastomas (GBM). Here, we highlight a precision medicine ... ...

Abstract	Li Fraumeni syndrome (LFS) is a hereditary cancer predisposition syndrome caused by germline mutations in TP53. TP53 is the most common mutated gene in human cancer, occurring in 30-50% of glioblastomas (GBM). Here, we highlight a precision medicine platform to identify potential targets for a GBM patient with LFS. We used a comparative transcriptomics approach to identify genes that are uniquely overexpressed in the LFS GBM patient relative to a cancer compendium of 12,747 tumor RNA sequencing data sets, including 200 GBMs. STAT1 and STAT2 were identified as being significantly overexpressed in the LFS patient, indicating ruxolitinib, a Janus kinase 1 and 2 inhibitors, as a potential therapy. The LFS patient had the highest level of STAT1 and STAT2 expression in an institutional high-grade glioma cohort of 45 patients, further supporting the cancer compendium results. To empirically validate the comparative transcriptomics pipeline, we used a combination of adherent and organoid cell culture techniques, including ex vivo patient-derived organoids (PDOs) from four patient-derived cell lines, including the LFS patient. STAT1 and STAT2 expression levels in the four patient-derived cells correlated with levels identified in the respective parent tumors. In both adherent and organoid cultures, cells from the LFS patient were among the most sensitive to ruxolitinib compared to patient-derived cells with lower STAT1 and STAT2 expression levels. A spheroid-based drug screening assay (3D-PREDICT) was performed and used to identify further therapeutic targets. Two targeted therapies were selected for the patient of interest and resulted in radiographic disease stability. This manuscript supports the use of comparative transcriptomics to identify personalized therapeutic targets in a functional precision medicine platform for malignant brain tumors.
MeSH term(s)	Adolescent ; Adult ; Child ; Female ; Gene Expression Regulation, Neoplastic ; Germ-Line Mutation/genetics ; Glioblastoma/complications ; Glioblastoma/genetics ; Glioblastoma/pathology ; Humans ; Janus Kinase 1/antagonists & inhibitors ; Janus Kinase 1/genetics ; Janus Kinase 2/antagonists & inhibitors ; Janus Kinase 2/genetics ; Li-Fraumeni Syndrome/complications ; Li-Fraumeni Syndrome/genetics ; Li-Fraumeni Syndrome/pathology ; Male ; Nitriles/pharmacology ; Organoids/metabolism ; Precision Medicine ; Pyrazoles/pharmacology ; Pyrimidines/pharmacology ; RNA-Seq ; STAT1 Transcription Factor/genetics ; STAT2 Transcription Factor/genetics ; Transcriptome/genetics ; Young Adult
Chemical Substances	Nitriles ; Pyrazoles ; Pyrimidines ; STAT1 Transcription Factor ; STAT1 protein, human ; STAT2 Transcription Factor ; STAT2 protein, human ; ruxolitinib (82S8X8XX8H) ; Janus Kinase 1 (EC 2.7.10.2) ; Janus Kinase 2 (EC 2.7.10.2)
Language	English
Publishing date	2021-12-02
Publishing country	Switzerland
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2661518-6
ISSN	2073-4409 ; 2073-4409
ISSN (online)	2073-4409
ISSN	2073-4409
DOI	10.3390/cells10123400
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Article: Human Translesion Polymerase Îº Exhibits Enhanced Activity and Reduced Fidelity Two Nucleotides from G-Quadruplex DNA

Eddy, Sarah / Tillman Magdalena / Maddukuri Leena / Ketkar Amit / Zafar Maroof K / Eoff Robert L

Biochemistry. 2016 Sept. 20, v. 55, no. 37

2016

Abstract: We have investigated the in vitro properties of human Y-family polymerase Îº (hpol Îº) on G-quadruplex DNA (G4 DNA). Similar to hpol Î·, another Y-family member implicated in replication of G4 motifs, hpol Îº bound G4 DNA with a 5.7-fold preference over ... ...

Abstract	We have investigated the in vitro properties of human Y-family polymerase Îº (hpol Îº) on G-quadruplex DNA (G4 DNA). Similar to hpol Î·, another Y-family member implicated in replication of G4 motifs, hpol Îº bound G4 DNA with a 5.7-fold preference over control, non-G4 DNA. Results from pol extension assays are consistent with the notion that G-quadruplexes present a stronger barrier to DNA synthesis by hpol Îº than they do to that by hpol Î·. However, kinetic analysis revealed that hpol Îº activity increases considerably when the enzyme is 2â3 nucleotides from the G4 motif, a trend that was reported previously for hpol Î·, though the increase was less pronounced. The increase in hpol Îº activity on G4 DNA was readily observed in the presence of either potassium or sodium but much less so when lithium was used in the buffer. The increased activity 2â3 nucleotides from the G4 motif was accompanied by a decrease in the fidelity of hpol Îº when the counterion was either potassium or sodium but not in the presence of lithium. The activity of hpol Îº decreased progressively as the primer was moved closer than 2 nucleotides from the G4 motif when either potassium or sodium was used to stabilize the G-quadruplex. Interestingly, the decrease in catalytic activity at the site of the quadruplex observed in potassium-containing buffer was accompanied by an increase in fidelity on G4 substrates versus control non-G4 substrates. This trend of increased fidelity in copying a tetrad-associated guanine was observed previously for hpol Î·, but not for the B-family member hpol Îµ, which exhibited a large decrease in both efficiency and fidelity in the attempt to copy the first guanine in the G4 motif. In summary, hpol Îº activity was enhanced relative to those of other Y-family members when the enzyme is 2â3 nucleotides from the G4 motif, but hpol Îº appears to be less competent than hpol Î· at copying tetrad-associated guanines.
Keywords	DNA ; catalytic activity ; guanine ; humans ; kinetics ; lithium ; nucleotides ; potassium ; sodium
Language	English
Dates of publication	2016-0920
Size	p. 5218-5229.
Publishing place	American Chemical Society
Document type	Article
ZDB-ID	1108-3
ISSN	1520-4995 ; 0006-2960
ISSN (online)	1520-4995
ISSN	0006-2960
DOI	10.1021%2Facs.biochem.6b00374
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Human Rev1 polymerase disrupts G-quadruplex DNA.

Eddy, Sarah / Ketkar, Amit / Zafar, Maroof K / Maddukuri, Leena / Choi, Jeong-Yun / Eoff, Robert L

Nucleic acids research

2013 Volume 42, Issue 5, Page(s) 3272–3285

Abstract: The Y-family DNA polymerase Rev1 is required for successful replication of G-quadruplex DNA (G4 DNA) in higher eukaryotes. Here we show that human Rev1 (hRev1) disrupts G4 DNA structures and prevents refolding in vitro. Nucleotidyl transfer by hRev1 is ... ...

Abstract	The Y-family DNA polymerase Rev1 is required for successful replication of G-quadruplex DNA (G4 DNA) in higher eukaryotes. Here we show that human Rev1 (hRev1) disrupts G4 DNA structures and prevents refolding in vitro. Nucleotidyl transfer by hRev1 is not necessary for mechanical unfolding to occur. hRev1 binds G4 DNA substrates with Kd,DNA values that are 4-15-fold lower than those of non-G4 DNA substrates. The pre-steady-state rate constant of deoxycytidine monophosphate (dCMP) insertion opposite the first tetrad-guanine by hRev1 is ∼56% as fast as that observed for non-G4 DNA substrates. Thus, hRev1 can promote fork progression by either dislodging tetrad guanines to unfold the G4 DNA, which could assist in extension by other DNA polymerases, or hRev1 can prevent refolding of G4 DNA structures. The hRev1 mechanism of action against G-quadruplexes helps explain why replication progress is impeded at G4 DNA sites in Rev1-deficient cells and illustrates another unique feature of this enzyme with important implications for genome maintenance.
MeSH term(s)	DNA/chemistry ; DNA/metabolism ; Deoxycytidine Monophosphate/metabolism ; G-Quadruplexes ; Guanine/chemistry ; Humans ; Nuclear Proteins/metabolism ; Nucleotidyltransferases/metabolism
Chemical Substances	Nuclear Proteins ; Deoxycytidine Monophosphate (1032-65-1) ; Guanine (5Z93L87A1R) ; DNA (9007-49-2) ; Nucleotidyltransferases (EC 2.7.7.-) ; REV1 protein, human (EC 2.7.7.-)
Language	English
Publishing date	2013-12-23
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	186809-3
ISSN	1362-4962 ; 1362-4954 ; 0301-5610 ; 0305-1048
ISSN (online)	1362-4962 ; 1362-4954
ISSN	0301-5610 ; 0305-1048
DOI	10.1093/nar/gkt1314
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Article ; Online: Evidence for the kinetic partitioning of polymerase activity on G-quadruplex DNA.

Eddy, Sarah / Maddukuri, Leena / Ketkar, Amit / Zafar, Maroof K / Henninger, Erin E / Pursell, Zachary F / Eoff, Robert L

Biochemistry

2015 Volume 54, Issue 20, Page(s) 3218–3230

Abstract: We have investigated the action of the human DNA polymerase ε (hpol ε) and η (hpol η) catalytic cores on G-quadruplex (G4) DNA substrates derived from the promoter of the c-MYC proto-oncogene. The translesion enzyme hpol η exhibits a 6.2-fold preference ... ...

Abstract	We have investigated the action of the human DNA polymerase ε (hpol ε) and η (hpol η) catalytic cores on G-quadruplex (G4) DNA substrates derived from the promoter of the c-MYC proto-oncogene. The translesion enzyme hpol η exhibits a 6.2-fold preference for binding to G4 DNA over non-G4 DNA, while hpol ε binds both G4 and non-G4 substrates with nearly equal affinity. Kinetic analysis of single-nucleotide insertion by hpol η reveals that it is able to maintain >25% activity on G4 substrates compared to non-G4 DNA substrates, even when the primer template junction is positioned directly adjacent to G22 (the first tetrad-associated guanine in the c-MYC G4 motif). Surprisingly, hpol η fidelity increases ~15-fold when copying G22. By way of comparison, hpol ε retains ~4% activity and has a 33-fold decrease in fidelity when copying G22. The fidelity of hpol η is ~100-fold greater than that of hpol ε when comparing the misinsertion frequencies of the two enzymes opposite a tetrad-associated guanine. The kinetic differences observed for the B- and Y-family pols on G4 DNA support a model in which a simple kinetic switch between replicative and TLS pols could help govern fork progress during G4 DNA replication.
MeSH term(s)	Base Pair Mismatch ; DNA Polymerase II/chemistry ; DNA Primers/chemistry ; DNA Replication ; DNA-Directed DNA Polymerase/chemistry ; G-Quadruplexes ; Humans ; Kinetics ; Protein Binding ; Substrate Specificity
Chemical Substances	DNA Primers ; DNA Polymerase II (EC 2.7.7.-) ; DNA-Directed DNA Polymerase (EC 2.7.7.7) ; Rad30 protein (EC 2.7.7.7)
Language	English
Publishing date	2015-05-26
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	1108-3
ISSN	1520-4995 ; 0006-2960
ISSN (online)	1520-4995
ISSN	0006-2960
DOI	10.1021/acs.biochem.5b00060
Database	MEDical Literature Analysis and Retrieval System OnLINE

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