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How a homolog of high-fidelity replicases conducts mutagenic DNA synthesis.

Lee YS, Gao Y, Yang W - Nat. Struct. Mol. Biol. (2015)

Bottom Line: A single-amino acid substitution in the O helix of the finger domain improves the fidelity of Pol ν nearly ten-fold.A unique cavity and the flexibility of the thumb domain allow Pol ν to generate and accommodate a looped-out primer strand.Primer loop-out may be a mechanism for DNA trinucloetide-repeat expansion.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA.

ABSTRACT
All DNA replicases achieve high fidelity by a conserved mechanism, but each translesion polymerase carries out mutagenic DNA synthesis in its own way. Here we report crystal structures of human DNA polymerase ν (Pol ν), which is homologous to high-fidelity replicases yet is error prone. Instead of a simple open-to-closed movement of the O helix upon binding of a correct incoming nucleotide, Pol ν has a different open state and requires the finger domain to swing sideways and undergo both opening and closing motions to accommodate the nascent base pair. A single-amino acid substitution in the O helix of the finger domain improves the fidelity of Pol ν nearly ten-fold. A unique cavity and the flexibility of the thumb domain allow Pol ν to generate and accommodate a looped-out primer strand. Primer loop-out may be a mechanism for DNA trinucloetide-repeat expansion.

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Diagram of DNA synthesis by Pol ν and a primer-loopout model for trinucleotide-repeat (TNR) expansion. (a) The high-fidelity Pol I and TLS Pol ν differ in the open states, while identical in the closed states. When the O helix in Pol ν is open for dNTP binding, helices Oa and Ob exclude the template base from the active site. The unique K679 in Pol ν further promotes dTTP misincorporation. The 3′–5′ exonuclease (Exo, pink star), which proofreads and improves the accuracy of Pol I, is inactivated in Pol ν (pseudo-Exo, black star). (b) A primer–loopout model. When a downstream template base is unusable (indicated as a red dot), Pol ν can loop out 1–2 nucleotides of the primer strand at the −3 position to re-use the normal template base(s) for lesion-bypass DNA synthesis. The mobile thumb of Pol ν (shown as semi-transparent green) may facilitate DNA translocation and misalignment. (c) Repetitive DNA sequence such as trinucleotide repeats (CNG)n would ease loopout of repeat units, as diagramed here, and result in repeat expansion.
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Figure 5: Diagram of DNA synthesis by Pol ν and a primer-loopout model for trinucleotide-repeat (TNR) expansion. (a) The high-fidelity Pol I and TLS Pol ν differ in the open states, while identical in the closed states. When the O helix in Pol ν is open for dNTP binding, helices Oa and Ob exclude the template base from the active site. The unique K679 in Pol ν further promotes dTTP misincorporation. The 3′–5′ exonuclease (Exo, pink star), which proofreads and improves the accuracy of Pol I, is inactivated in Pol ν (pseudo-Exo, black star). (b) A primer–loopout model. When a downstream template base is unusable (indicated as a red dot), Pol ν can loop out 1–2 nucleotides of the primer strand at the −3 position to re-use the normal template base(s) for lesion-bypass DNA synthesis. The mobile thumb of Pol ν (shown as semi-transparent green) may facilitate DNA translocation and misalignment. (c) Repetitive DNA sequence such as trinucleotide repeats (CNG)n would ease loopout of repeat units, as diagramed here, and result in repeat expansion.

Mentions: Until now a simple open and closed rotation of the finger domain during each cycle of nucleotide incorporation has been universally observed in A-, B-, C-, and X-family DNA polymerases 16,18,28,29, reverse transcriptases, and RNA polymerases 37,38 (Fig. 5a). The Pol ν75 structures, however, reveal an unprecedented open state, which is open for an incoming nucleotide, but closed for template base binding. The unusual open state would change the kinetic and dynamic processes of nascent base pair formation, incoming nucleotide selection, and incorporation. A handful of residues unique to Pol ν (Fig. 3a) likely stabilize the unusual open state, and conservation of Y682 and G689 in Pol ν and Pol θ (Fig. 3a) indicates that the unusual open state is likely also present in Pol θ.


How a homolog of high-fidelity replicases conducts mutagenic DNA synthesis.

Lee YS, Gao Y, Yang W - Nat. Struct. Mol. Biol. (2015)

Diagram of DNA synthesis by Pol ν and a primer-loopout model for trinucleotide-repeat (TNR) expansion. (a) The high-fidelity Pol I and TLS Pol ν differ in the open states, while identical in the closed states. When the O helix in Pol ν is open for dNTP binding, helices Oa and Ob exclude the template base from the active site. The unique K679 in Pol ν further promotes dTTP misincorporation. The 3′–5′ exonuclease (Exo, pink star), which proofreads and improves the accuracy of Pol I, is inactivated in Pol ν (pseudo-Exo, black star). (b) A primer–loopout model. When a downstream template base is unusable (indicated as a red dot), Pol ν can loop out 1–2 nucleotides of the primer strand at the −3 position to re-use the normal template base(s) for lesion-bypass DNA synthesis. The mobile thumb of Pol ν (shown as semi-transparent green) may facilitate DNA translocation and misalignment. (c) Repetitive DNA sequence such as trinucleotide repeats (CNG)n would ease loopout of repeat units, as diagramed here, and result in repeat expansion.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4469489&req=5

Figure 5: Diagram of DNA synthesis by Pol ν and a primer-loopout model for trinucleotide-repeat (TNR) expansion. (a) The high-fidelity Pol I and TLS Pol ν differ in the open states, while identical in the closed states. When the O helix in Pol ν is open for dNTP binding, helices Oa and Ob exclude the template base from the active site. The unique K679 in Pol ν further promotes dTTP misincorporation. The 3′–5′ exonuclease (Exo, pink star), which proofreads and improves the accuracy of Pol I, is inactivated in Pol ν (pseudo-Exo, black star). (b) A primer–loopout model. When a downstream template base is unusable (indicated as a red dot), Pol ν can loop out 1–2 nucleotides of the primer strand at the −3 position to re-use the normal template base(s) for lesion-bypass DNA synthesis. The mobile thumb of Pol ν (shown as semi-transparent green) may facilitate DNA translocation and misalignment. (c) Repetitive DNA sequence such as trinucleotide repeats (CNG)n would ease loopout of repeat units, as diagramed here, and result in repeat expansion.
Mentions: Until now a simple open and closed rotation of the finger domain during each cycle of nucleotide incorporation has been universally observed in A-, B-, C-, and X-family DNA polymerases 16,18,28,29, reverse transcriptases, and RNA polymerases 37,38 (Fig. 5a). The Pol ν75 structures, however, reveal an unprecedented open state, which is open for an incoming nucleotide, but closed for template base binding. The unusual open state would change the kinetic and dynamic processes of nascent base pair formation, incoming nucleotide selection, and incorporation. A handful of residues unique to Pol ν (Fig. 3a) likely stabilize the unusual open state, and conservation of Y682 and G689 in Pol ν and Pol θ (Fig. 3a) indicates that the unusual open state is likely also present in Pol θ.

Bottom Line: A single-amino acid substitution in the O helix of the finger domain improves the fidelity of Pol ν nearly ten-fold.A unique cavity and the flexibility of the thumb domain allow Pol ν to generate and accommodate a looped-out primer strand.Primer loop-out may be a mechanism for DNA trinucloetide-repeat expansion.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA.

ABSTRACT
All DNA replicases achieve high fidelity by a conserved mechanism, but each translesion polymerase carries out mutagenic DNA synthesis in its own way. Here we report crystal structures of human DNA polymerase ν (Pol ν), which is homologous to high-fidelity replicases yet is error prone. Instead of a simple open-to-closed movement of the O helix upon binding of a correct incoming nucleotide, Pol ν has a different open state and requires the finger domain to swing sideways and undergo both opening and closing motions to accommodate the nascent base pair. A single-amino acid substitution in the O helix of the finger domain improves the fidelity of Pol ν nearly ten-fold. A unique cavity and the flexibility of the thumb domain allow Pol ν to generate and accommodate a looped-out primer strand. Primer loop-out may be a mechanism for DNA trinucloetide-repeat expansion.

Show MeSH
Related in: MedlinePlus