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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 a primer-template containing a thymine–thymine 6–4 photoproduct dimer (6-4pp) (Figure 2D; ZZ = dimer) by Tgo-Pol and Tgo-Pol Z1. (A1) and (A2) Products seen using the primer that initiates synthesis immediately before the lesion with all four dNTPs (A1) or a single dNTP (A2). (B1) and (B2) Products observed with the primer that begins synthesis at the first (3′) base in the 6–4 photoproduct dimer, with the last (3′) base in the primer being either dC (B1) or dA (B2). (C) Products produced with the primer that commences polymerisation at the second (5′) base of the lesion. (D) Products seen using the primer that begins synthesis at the standard dC base that immediately follows the lesion.
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Figure 6: Extension of a primer-template containing a thymine–thymine 6–4 photoproduct dimer (6-4pp) (Figure 2D; ZZ = dimer) by Tgo-Pol and Tgo-Pol Z1. (A1) and (A2) Products seen using the primer that initiates synthesis immediately before the lesion with all four dNTPs (A1) or a single dNTP (A2). (B1) and (B2) Products observed with the primer that begins synthesis at the first (3′) base in the 6–4 photoproduct dimer, with the last (3′) base in the primer being either dC (B1) or dA (B2). (C) Products produced with the primer that commences polymerisation at the second (5′) base of the lesion. (D) Products seen using the primer that begins synthesis at the standard dC base that immediately follows the lesion.

Mentions: The thymine–thymine 6–4 photoproduct (6–4pp) was a more powerful block to both Tgo-Pol and Tgo-Pol Z1 than the CPD lesion. Due to the difficulties in incorporating the 6–4pp lesion into oligodeoxynucleotides, a short template had to be used and, as a consequence, one primer initiated synthesis from immediately before the lesion (a running start primer would not be long enough to produce a stable duplex) (Figure 2D). With both polymerases, the addition of all four dNTPs resulted in slow addition of one base, followed by a less efficient second incorporation (Figure 6A). With Tgo-Pol Z1, noticeable addition of a third base was also observed. Single dNTP incorporation showed Tgo-Pol preferentially incorporated purines across the first lesion base, although weaker activity with pyrimidines was also apparent. With Tgo-Pol Z1, a similar selectivity was observed, although incorporation of dTMP was more prominent than for the wild-type enzyme. For both enzymes, only dATP led to slow incorporation of two bases to completely cover the 6–4pp lesion (Figure 6A). With a primer that terminated opposite the first (3′) lesion base, further extension was only observed when the deoxynucleotide at the 3′ primer end (the initiation point for synthesis) was dC or dA (Figure 6B). When dC was 3′ terminal base of the primer, Tgo-Pol slowly added a single base; Tgo-Pol Z1 more rapidly incorporated one base and could slowly catalyse further extension. More pronounced activity was observed with dA as the initiating deoxynucleotide. Tgo-Pol introduced one base and, more slowly, a second; Tgo-Pol Z1 incorporated the first base at about the same rate as Tgo-Pol, but was much quicker at adding a second base and also incorporated further deoxynucleotides (Figure 6B). With dG or dT as the 3′ base with these primers, very little incorporation was seen with either polymerase (data not shown). Using a primer that placed two adenines (dAA) across the 6–4pp lesion led to near complete inhibition of Tgo-Pol; in contrast, Tgo-Pol Z1 was able to continue polymerisation (Figure 6C). Finally, very similar results were observed with the longest primer, which covers both the 6–4pp lesion and the first post damage base. Tgo-Pol was unable to extend from this primer whereas Tgo-Pol Z1 showed clear primer extension (Figure 6D).


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 a primer-template containing a thymine–thymine 6–4 photoproduct dimer (6-4pp) (Figure 2D; ZZ = dimer) by Tgo-Pol and Tgo-Pol Z1. (A1) and (A2) Products seen using the primer that initiates synthesis immediately before the lesion with all four dNTPs (A1) or a single dNTP (A2). (B1) and (B2) Products observed with the primer that begins synthesis at the first (3′) base in the 6–4 photoproduct dimer, with the last (3′) base in the primer being either dC (B1) or dA (B2). (C) Products produced with the primer that commences polymerisation at the second (5′) base of the lesion. (D) Products seen using the primer that begins synthesis at the standard dC base that immediately follows the lesion.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4150786&req=5

Figure 6: Extension of a primer-template containing a thymine–thymine 6–4 photoproduct dimer (6-4pp) (Figure 2D; ZZ = dimer) by Tgo-Pol and Tgo-Pol Z1. (A1) and (A2) Products seen using the primer that initiates synthesis immediately before the lesion with all four dNTPs (A1) or a single dNTP (A2). (B1) and (B2) Products observed with the primer that begins synthesis at the first (3′) base in the 6–4 photoproduct dimer, with the last (3′) base in the primer being either dC (B1) or dA (B2). (C) Products produced with the primer that commences polymerisation at the second (5′) base of the lesion. (D) Products seen using the primer that begins synthesis at the standard dC base that immediately follows the lesion.
Mentions: The thymine–thymine 6–4 photoproduct (6–4pp) was a more powerful block to both Tgo-Pol and Tgo-Pol Z1 than the CPD lesion. Due to the difficulties in incorporating the 6–4pp lesion into oligodeoxynucleotides, a short template had to be used and, as a consequence, one primer initiated synthesis from immediately before the lesion (a running start primer would not be long enough to produce a stable duplex) (Figure 2D). With both polymerases, the addition of all four dNTPs resulted in slow addition of one base, followed by a less efficient second incorporation (Figure 6A). With Tgo-Pol Z1, noticeable addition of a third base was also observed. Single dNTP incorporation showed Tgo-Pol preferentially incorporated purines across the first lesion base, although weaker activity with pyrimidines was also apparent. With Tgo-Pol Z1, a similar selectivity was observed, although incorporation of dTMP was more prominent than for the wild-type enzyme. For both enzymes, only dATP led to slow incorporation of two bases to completely cover the 6–4pp lesion (Figure 6A). With a primer that terminated opposite the first (3′) lesion base, further extension was only observed when the deoxynucleotide at the 3′ primer end (the initiation point for synthesis) was dC or dA (Figure 6B). When dC was 3′ terminal base of the primer, Tgo-Pol slowly added a single base; Tgo-Pol Z1 more rapidly incorporated one base and could slowly catalyse further extension. More pronounced activity was observed with dA as the initiating deoxynucleotide. Tgo-Pol introduced one base and, more slowly, a second; Tgo-Pol Z1 incorporated the first base at about the same rate as Tgo-Pol, but was much quicker at adding a second base and also incorporated further deoxynucleotides (Figure 6B). With dG or dT as the 3′ base with these primers, very little incorporation was seen with either polymerase (data not shown). Using a primer that placed two adenines (dAA) across the 6–4pp lesion led to near complete inhibition of Tgo-Pol; in contrast, Tgo-Pol Z1 was able to continue polymerisation (Figure 6C). Finally, very similar results were observed with the longest primer, which covers both the 6–4pp lesion and the first post damage base. Tgo-Pol was unable to extend from this primer whereas Tgo-Pol Z1 showed clear primer extension (Figure 6D).

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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Related in: MedlinePlus