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An archaeal family-B DNA polymerase variant able to replicate past DNA damage: occurrence of replicative and translesion synthesis polymerases within the B family.

Jozwiakowski SK, Keith BJ, Gilroy L, Doherty AJ, Connolly BA - Nucleic Acids Res. (2014)

Bottom Line: The resulting Tgo-Pol Z1 variant is proficient at initiating replication from base mismatches and can read through damaged bases, such as abasic sites and thymine photo-dimers.The fidelity of Tgo-Pol Z1 is reduced, with a marked tendency to make changes at G:C base pairs.Tgo-Pol Z1 may also be useful for amplification of damaged DNA.

View Article: PubMed Central - PubMed

Affiliation: Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton BN1 9RQ, UK Institute of Cell and Molecular Biosciences, University of Newcastle, Newcastle upon Tyne, NE2 4HH, UK s.k.jozwiakowski@sussex.ac.uk.

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Extension of control DNA and primer-templates that terminate in base mismatches. (A) Polymerisation of a control DNA lacking modified bases by Tgo-Pol and Tgo-Pol Z1. The primer-template shown in Figure 2A (X = T, Y = T; running start primer) was used. The main panel (A1) shows extension with all four dNTPs added. The smaller panel (A2) shows what takes place when only a single dNTP is used (with this primer-template combination, the first and second template bases are both dC; therefore, addition of two dGTPs represents ‘correct’ insertion). (B) Extension of a primer-template from a C:T base mismatch (Figure 2B) by Tgo-Pol and Tgo-Pol Z1. (C) Extension of a primer-template from a T:T base mismatch (Figure 2C) by Tgo-Pol and Tgo-Pol Z1.
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Figure 3: Extension of control DNA and primer-templates that terminate in base mismatches. (A) Polymerisation of a control DNA lacking modified bases by Tgo-Pol and Tgo-Pol Z1. The primer-template shown in Figure 2A (X = T, Y = T; running start primer) was used. The main panel (A1) shows extension with all four dNTPs added. The smaller panel (A2) shows what takes place when only a single dNTP is used (with this primer-template combination, the first and second template bases are both dC; therefore, addition of two dGTPs represents ‘correct’ insertion). (B) Extension of a primer-template from a C:T base mismatch (Figure 2B) by Tgo-Pol and Tgo-Pol Z1. (C) Extension of a primer-template from a T:T base mismatch (Figure 2C) by Tgo-Pol and Tgo-Pol Z1.

Mentions: Little difference in activity was observed between Tgo-Pol and Tgo-Pol Z1 (both derivatives are exo−) when a standard primer-template was extended, suggesting that altering the fingers loop has little influence on intrinsic activity (Figure 3A). Notably, Tgo-Pol Z1 seems to show slightly lower activity than the parental Tgo-Pol when the primer extension rates were compared. Confirmation came from the near identical performance of the two proteins in PCR reactions (Supplementary Figure S1). Similarly, only minor differences were observed when just a single dNTP was used for extension. Using a template with two cytosine bases immediately ahead of 3′ end of the primer, both polymerases rapidly incorporated, as expected, two dGMPs (Figure 3A). But mis-incorporation of a single dAMP and dTMP (and to a much lesser extent dCMP) was also apparent. Tgo-Pol Z1 may be marginally better at mis-incorporation as shown by the slightly more pronounced bands at +4 in the dG lane and +2 and +3 in the dT lane. With a fully ‘intact’ polymerase, such mismatched bases would normally be excised by the 3′–5′ proof-reading exonuclease activity (42). To further investigate the ability of the polymerases to extend mis-pairs, we used two primer-templates that direct replication from either a mismatched C:T or T:T base pair (Figure 2B and C). Usually, such mistakes occur when a polymerase pairs a template base with an incorrect dNTP during replication. If such errors escape correction by the proof-reading exonuclease activity of the replicating polymerase, subsequent genome copying must commence from the resulting mismatch. Differences between Tgo-Pol and Tgo-Pol Z1 were readily apparent, with Tgo-Pol Z1 extending the mismatched primers noticeably more rapidly than the control with both primer-templates (Figure 3B and C). Thus, the insertion of the Pol ζ fingers domain into Tgo-Pol results in a derivative better able to extend from DNA base mismatches.


An archaeal family-B DNA polymerase variant able to replicate past DNA damage: occurrence of replicative and translesion synthesis polymerases within the B family.

Jozwiakowski SK, Keith BJ, Gilroy L, Doherty AJ, Connolly BA - Nucleic Acids Res. (2014)

Extension of control DNA and primer-templates that terminate in base mismatches. (A) Polymerisation of a control DNA lacking modified bases by Tgo-Pol and Tgo-Pol Z1. The primer-template shown in Figure 2A (X = T, Y = T; running start primer) was used. The main panel (A1) shows extension with all four dNTPs added. The smaller panel (A2) shows what takes place when only a single dNTP is used (with this primer-template combination, the first and second template bases are both dC; therefore, addition of two dGTPs represents ‘correct’ insertion). (B) Extension of a primer-template from a C:T base mismatch (Figure 2B) by Tgo-Pol and Tgo-Pol Z1. (C) Extension of a primer-template from a T:T base mismatch (Figure 2C) by Tgo-Pol and Tgo-Pol Z1.
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Figure 3: Extension of control DNA and primer-templates that terminate in base mismatches. (A) Polymerisation of a control DNA lacking modified bases by Tgo-Pol and Tgo-Pol Z1. The primer-template shown in Figure 2A (X = T, Y = T; running start primer) was used. The main panel (A1) shows extension with all four dNTPs added. The smaller panel (A2) shows what takes place when only a single dNTP is used (with this primer-template combination, the first and second template bases are both dC; therefore, addition of two dGTPs represents ‘correct’ insertion). (B) Extension of a primer-template from a C:T base mismatch (Figure 2B) by Tgo-Pol and Tgo-Pol Z1. (C) Extension of a primer-template from a T:T base mismatch (Figure 2C) by Tgo-Pol and Tgo-Pol Z1.
Mentions: Little difference in activity was observed between Tgo-Pol and Tgo-Pol Z1 (both derivatives are exo−) when a standard primer-template was extended, suggesting that altering the fingers loop has little influence on intrinsic activity (Figure 3A). Notably, Tgo-Pol Z1 seems to show slightly lower activity than the parental Tgo-Pol when the primer extension rates were compared. Confirmation came from the near identical performance of the two proteins in PCR reactions (Supplementary Figure S1). Similarly, only minor differences were observed when just a single dNTP was used for extension. Using a template with two cytosine bases immediately ahead of 3′ end of the primer, both polymerases rapidly incorporated, as expected, two dGMPs (Figure 3A). But mis-incorporation of a single dAMP and dTMP (and to a much lesser extent dCMP) was also apparent. Tgo-Pol Z1 may be marginally better at mis-incorporation as shown by the slightly more pronounced bands at +4 in the dG lane and +2 and +3 in the dT lane. With a fully ‘intact’ polymerase, such mismatched bases would normally be excised by the 3′–5′ proof-reading exonuclease activity (42). To further investigate the ability of the polymerases to extend mis-pairs, we used two primer-templates that direct replication from either a mismatched C:T or T:T base pair (Figure 2B and C). Usually, such mistakes occur when a polymerase pairs a template base with an incorrect dNTP during replication. If such errors escape correction by the proof-reading exonuclease activity of the replicating polymerase, subsequent genome copying must commence from the resulting mismatch. Differences between Tgo-Pol and Tgo-Pol Z1 were readily apparent, with Tgo-Pol Z1 extending the mismatched primers noticeably more rapidly than the control with both primer-templates (Figure 3B and C). Thus, the insertion of the Pol ζ fingers domain into Tgo-Pol results in a derivative better able to extend from DNA base mismatches.

Bottom Line: The resulting Tgo-Pol Z1 variant is proficient at initiating replication from base mismatches and can read through damaged bases, such as abasic sites and thymine photo-dimers.The fidelity of Tgo-Pol Z1 is reduced, with a marked tendency to make changes at G:C base pairs.Tgo-Pol Z1 may also be useful for amplification of damaged DNA.

View Article: PubMed Central - PubMed

Affiliation: Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton BN1 9RQ, UK Institute of Cell and Molecular Biosciences, University of Newcastle, Newcastle upon Tyne, NE2 4HH, UK s.k.jozwiakowski@sussex.ac.uk.

Show MeSH
Related in: MedlinePlus