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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 cis-syn thymine–thymine cyclobutane pyrimidine dimer (CPD) (Figure 2A; XY = dimer) by Tgo-Pol and Tgo-Pol Z1. (A1) Products produced using the running start primer in the presence of all four dNTPs. (A2) Products observed using a single dNTP and the primers that initiate synthesis immediately before the lesion (A2a) or opposite the first (3′) base of the lesion (A2b). (A3) Products found with the primer that commences polymerisation at the second (5′) base of the thymine dimer in the presence of all four dNTPs.
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Figure 5: Extension of a primer-template containing a cis-syn thymine–thymine cyclobutane pyrimidine dimer (CPD) (Figure 2A; XY = dimer) by Tgo-Pol and Tgo-Pol Z1. (A1) Products produced using the running start primer in the presence of all four dNTPs. (A2) Products observed using a single dNTP and the primers that initiate synthesis immediately before the lesion (A2a) or opposite the first (3′) base of the lesion (A2b). (A3) Products found with the primer that commences polymerisation at the second (5′) base of the thymine dimer in the presence of all four dNTPs.

Mentions: Much damage to DNA, e.g. abasic sites and thymine glycol discussed above, requires a polymerase to traverse a single lesion. Exposure of DNA to UV radiation can give rise to the thymine photodimers, the most abundant of which are the cyclobutane pyrimidine dimer (CPD) and the 6–4 photoproduct (6–4pp) (46,47). In these cases, the polymerase has a more demanding task in that it must negotiate two damaged bases. The presence of a thymine–thymine cis-syn cyclobutane dimer essentially blocks passage by wild-type Tgo-Pol (Figure 5). With the running start primer, a strong stop was observed at +7 (immediately before the CPD) and a slightly weaker one at +8 (corresponding to copying the first base in the CPD). Further investigation made use of three primers that commence polymerisation from either just before the CPD or at the first or second damaged T within the lesion (Figure 2A). Tgo-Pol was capable of adding a purine opposite the initial lesion base; however, no incorporation at the second position took place and extension from the double mismatch between the templating CPD and two adenines (dAA) of the primer 3′ end was also not possible (Figure 5). The single base incorporations are fully compatible with the running primers results, which indicate pausing just before the CPD and at the first damaged thymine of the lesion. In contrast to the wild type, Tgo-Pol Z1 is able to read through the CPD and full-length product is slowly formed with the running start primer (Figure 5). Using the three primers designed to initiate synthesis at the lesion (Figure 2A) showed that Tgo-Pol Z1 could incorporate dAMP, dGMP or dTMP across the first (3′) damaged thymine and dAMP opposite the second (5′) thymine of the CPD. Additionally, extension from double mismatch between templating CPD lesion and two adenines (dAA) located at 3′ end of the primer was observed in case of Tgo-Pol Z1 (Figure 5).


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 cis-syn thymine–thymine cyclobutane pyrimidine dimer (CPD) (Figure 2A; XY = dimer) by Tgo-Pol and Tgo-Pol Z1. (A1) Products produced using the running start primer in the presence of all four dNTPs. (A2) Products observed using a single dNTP and the primers that initiate synthesis immediately before the lesion (A2a) or opposite the first (3′) base of the lesion (A2b). (A3) Products found with the primer that commences polymerisation at the second (5′) base of the thymine dimer in the presence of all four dNTPs.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 5: Extension of a primer-template containing a cis-syn thymine–thymine cyclobutane pyrimidine dimer (CPD) (Figure 2A; XY = dimer) by Tgo-Pol and Tgo-Pol Z1. (A1) Products produced using the running start primer in the presence of all four dNTPs. (A2) Products observed using a single dNTP and the primers that initiate synthesis immediately before the lesion (A2a) or opposite the first (3′) base of the lesion (A2b). (A3) Products found with the primer that commences polymerisation at the second (5′) base of the thymine dimer in the presence of all four dNTPs.
Mentions: Much damage to DNA, e.g. abasic sites and thymine glycol discussed above, requires a polymerase to traverse a single lesion. Exposure of DNA to UV radiation can give rise to the thymine photodimers, the most abundant of which are the cyclobutane pyrimidine dimer (CPD) and the 6–4 photoproduct (6–4pp) (46,47). In these cases, the polymerase has a more demanding task in that it must negotiate two damaged bases. The presence of a thymine–thymine cis-syn cyclobutane dimer essentially blocks passage by wild-type Tgo-Pol (Figure 5). With the running start primer, a strong stop was observed at +7 (immediately before the CPD) and a slightly weaker one at +8 (corresponding to copying the first base in the CPD). Further investigation made use of three primers that commence polymerisation from either just before the CPD or at the first or second damaged T within the lesion (Figure 2A). Tgo-Pol was capable of adding a purine opposite the initial lesion base; however, no incorporation at the second position took place and extension from the double mismatch between the templating CPD and two adenines (dAA) of the primer 3′ end was also not possible (Figure 5). The single base incorporations are fully compatible with the running primers results, which indicate pausing just before the CPD and at the first damaged thymine of the lesion. In contrast to the wild type, Tgo-Pol Z1 is able to read through the CPD and full-length product is slowly formed with the running start primer (Figure 5). Using the three primers designed to initiate synthesis at the lesion (Figure 2A) showed that Tgo-Pol Z1 could incorporate dAMP, dGMP or dTMP across the first (3′) damaged thymine and dAMP opposite the second (5′) thymine of the CPD. Additionally, extension from double mismatch between templating CPD lesion and two adenines (dAA) located at 3′ end of the primer was observed in case of Tgo-Pol Z1 (Figure 5).

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