Kinetic analysis of human PrimPol DNA polymerase activity reveals a generally error-prone enzyme capable of accurately bypassing 7,8-dihydro-8-oxo-2′-deoxyguanosine

Maroof K. Zafar, Amit Ketkar, Maria F. Lodeiro, Craig E. Cameron, Robert L. Eoff

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

Recent studies have identified human PrimPol as a new RNA/DNA primase and translesion DNA synthesis polymerase (TLS pol) that contributes to nuclear and mitochondrial DNA replication. We investigated the mechanism of PrimPol polymerase activity on both undamaged and damaged DNA substrates. With Mg2+ as a cofactor, PrimPol binds primer-template DNA with low affinity Kd,DNA values (∼200-1200 nM). DNA binding is enhanced 34-fold by Mn2+ (Kd,DNA = 27 nM). The pol activity of PrimPol is increased 400-1000-fold by Mn2+ compared to Mg2+ based on steady-state kinetic parameters. PrimPol makes a mistake copying undamaged DNA once every ∼100-2500 insertions events, which is comparable to other TLS pols, and the fidelity of PrimPol is ∼1.7-fold more accurate when Mg2+ is the cofactor compared to Mn2+. PrimPol inserts dCMP opposite 8-oxo-dG with 2- (Mn2+) to 6-fold (Mg2+) greater efficiency than dAMP misinsertion. PrimPol-catalyzed dCMP insertion opposite 8-oxo-dG proceeds at ∼25% efficiency relative to unmodified template dG, and PrimPol readily extends from dC:8-oxo-dG base pairs (bps) with ∼2-fold greater efficiency than dA:8-oxo-dG bps. A tetrahydrofuran (THF) abasic-site mimic decreases PrimPol activity to ∼0.04%. In summary, PrimPol exhibits the fidelity typical of other TLS pols, is rather unusual in the degree of activation afforded by Mn2+, and accurately bypasses 8-oxo-dG, a DNA lesion of special relevance to mitochondrial DNA replication and transcription.

Original languageEnglish (US)
Pages (from-to)6584-6594
Number of pages11
JournalBiochemistry
Volume53
Issue number41
DOIs
StatePublished - Oct 21 2014

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DNA-Directed DNA Polymerase
Kinetics
DNA
Enzymes
DNA Replication
Mitochondrial DNA
Base Pairing
DNA Primase
DNA Primers
Copying
8-oxo-7-hydrodeoxyguanosine
Transcription
Kinetic parameters
RNA
Chemical activation
Substrates

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Zafar, Maroof K. ; Ketkar, Amit ; Lodeiro, Maria F. ; Cameron, Craig E. ; Eoff, Robert L. / Kinetic analysis of human PrimPol DNA polymerase activity reveals a generally error-prone enzyme capable of accurately bypassing 7,8-dihydro-8-oxo-2′-deoxyguanosine. In: Biochemistry. 2014 ; Vol. 53, No. 41. pp. 6584-6594.
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abstract = "Recent studies have identified human PrimPol as a new RNA/DNA primase and translesion DNA synthesis polymerase (TLS pol) that contributes to nuclear and mitochondrial DNA replication. We investigated the mechanism of PrimPol polymerase activity on both undamaged and damaged DNA substrates. With Mg2+ as a cofactor, PrimPol binds primer-template DNA with low affinity Kd,DNA values (∼200-1200 nM). DNA binding is enhanced 34-fold by Mn2+ (Kd,DNA = 27 nM). The pol activity of PrimPol is increased 400-1000-fold by Mn2+ compared to Mg2+ based on steady-state kinetic parameters. PrimPol makes a mistake copying undamaged DNA once every ∼100-2500 insertions events, which is comparable to other TLS pols, and the fidelity of PrimPol is ∼1.7-fold more accurate when Mg2+ is the cofactor compared to Mn2+. PrimPol inserts dCMP opposite 8-oxo-dG with 2- (Mn2+) to 6-fold (Mg2+) greater efficiency than dAMP misinsertion. PrimPol-catalyzed dCMP insertion opposite 8-oxo-dG proceeds at ∼25{\%} efficiency relative to unmodified template dG, and PrimPol readily extends from dC:8-oxo-dG base pairs (bps) with ∼2-fold greater efficiency than dA:8-oxo-dG bps. A tetrahydrofuran (THF) abasic-site mimic decreases PrimPol activity to ∼0.04{\%}. In summary, PrimPol exhibits the fidelity typical of other TLS pols, is rather unusual in the degree of activation afforded by Mn2+, and accurately bypasses 8-oxo-dG, a DNA lesion of special relevance to mitochondrial DNA replication and transcription.",
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Kinetic analysis of human PrimPol DNA polymerase activity reveals a generally error-prone enzyme capable of accurately bypassing 7,8-dihydro-8-oxo-2′-deoxyguanosine. / Zafar, Maroof K.; Ketkar, Amit; Lodeiro, Maria F.; Cameron, Craig E.; Eoff, Robert L.

In: Biochemistry, Vol. 53, No. 41, 21.10.2014, p. 6584-6594.

Research output: Contribution to journalArticle

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T1 - Kinetic analysis of human PrimPol DNA polymerase activity reveals a generally error-prone enzyme capable of accurately bypassing 7,8-dihydro-8-oxo-2′-deoxyguanosine

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AU - Ketkar, Amit

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N2 - Recent studies have identified human PrimPol as a new RNA/DNA primase and translesion DNA synthesis polymerase (TLS pol) that contributes to nuclear and mitochondrial DNA replication. We investigated the mechanism of PrimPol polymerase activity on both undamaged and damaged DNA substrates. With Mg2+ as a cofactor, PrimPol binds primer-template DNA with low affinity Kd,DNA values (∼200-1200 nM). DNA binding is enhanced 34-fold by Mn2+ (Kd,DNA = 27 nM). The pol activity of PrimPol is increased 400-1000-fold by Mn2+ compared to Mg2+ based on steady-state kinetic parameters. PrimPol makes a mistake copying undamaged DNA once every ∼100-2500 insertions events, which is comparable to other TLS pols, and the fidelity of PrimPol is ∼1.7-fold more accurate when Mg2+ is the cofactor compared to Mn2+. PrimPol inserts dCMP opposite 8-oxo-dG with 2- (Mn2+) to 6-fold (Mg2+) greater efficiency than dAMP misinsertion. PrimPol-catalyzed dCMP insertion opposite 8-oxo-dG proceeds at ∼25% efficiency relative to unmodified template dG, and PrimPol readily extends from dC:8-oxo-dG base pairs (bps) with ∼2-fold greater efficiency than dA:8-oxo-dG bps. A tetrahydrofuran (THF) abasic-site mimic decreases PrimPol activity to ∼0.04%. In summary, PrimPol exhibits the fidelity typical of other TLS pols, is rather unusual in the degree of activation afforded by Mn2+, and accurately bypasses 8-oxo-dG, a DNA lesion of special relevance to mitochondrial DNA replication and transcription.

AB - Recent studies have identified human PrimPol as a new RNA/DNA primase and translesion DNA synthesis polymerase (TLS pol) that contributes to nuclear and mitochondrial DNA replication. We investigated the mechanism of PrimPol polymerase activity on both undamaged and damaged DNA substrates. With Mg2+ as a cofactor, PrimPol binds primer-template DNA with low affinity Kd,DNA values (∼200-1200 nM). DNA binding is enhanced 34-fold by Mn2+ (Kd,DNA = 27 nM). The pol activity of PrimPol is increased 400-1000-fold by Mn2+ compared to Mg2+ based on steady-state kinetic parameters. PrimPol makes a mistake copying undamaged DNA once every ∼100-2500 insertions events, which is comparable to other TLS pols, and the fidelity of PrimPol is ∼1.7-fold more accurate when Mg2+ is the cofactor compared to Mn2+. PrimPol inserts dCMP opposite 8-oxo-dG with 2- (Mn2+) to 6-fold (Mg2+) greater efficiency than dAMP misinsertion. PrimPol-catalyzed dCMP insertion opposite 8-oxo-dG proceeds at ∼25% efficiency relative to unmodified template dG, and PrimPol readily extends from dC:8-oxo-dG base pairs (bps) with ∼2-fold greater efficiency than dA:8-oxo-dG bps. A tetrahydrofuran (THF) abasic-site mimic decreases PrimPol activity to ∼0.04%. In summary, PrimPol exhibits the fidelity typical of other TLS pols, is rather unusual in the degree of activation afforded by Mn2+, and accurately bypasses 8-oxo-dG, a DNA lesion of special relevance to mitochondrial DNA replication and transcription.

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