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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 primer-templates that contain a single damaged base in the template. (A) Copying of a template containing an abasic site (Ab) (Figure 2A; X = T, Y = abasic site) by Tgo-Pol and Tgo-Pol Z1. (A1) shows the results found with the running start primer and all four dNTPs. (A2) uses the primer that initiates polymerisation from just before the abasic site (Figure 2A) and only a single dNTP to determine preferential incorporation opposite the lesion. (A3) utilises a primer that has a dA base across from the abasic site (Figure 2A), enabling extension from the dA:abasic site mis-pair to be evaluated. (B) Copying of a template containing a thymine glycol (Thgl) (Figure 2A; X = T, Y = thymine glycol) by Tgo-Pol and Tgo-Pol Z1. (B1), (B2) and (B3) are identical to A1, A2 and A3, except a thymine glycol replaces the abasic site.
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Figure 4: Extension of primer-templates that contain a single damaged base in the template. (A) Copying of a template containing an abasic site (Ab) (Figure 2A; X = T, Y = abasic site) by Tgo-Pol and Tgo-Pol Z1. (A1) shows the results found with the running start primer and all four dNTPs. (A2) uses the primer that initiates polymerisation from just before the abasic site (Figure 2A) and only a single dNTP to determine preferential incorporation opposite the lesion. (A3) utilises a primer that has a dA base across from the abasic site (Figure 2A), enabling extension from the dA:abasic site mis-pair to be evaluated. (B) Copying of a template containing a thymine glycol (Thgl) (Figure 2A; X = T, Y = thymine glycol) by Tgo-Pol and Tgo-Pol Z1. (B1), (B2) and (B3) are identical to A1, A2 and A3, except a thymine glycol replaces the abasic site.

Mentions: To investigate the effects of introducing a damaged base into DNA, two types of lesions, an abasic site and a thymine glycol, were selected. Abasic sites can arise spontaneously by depurination/depyrimidation of both standard and damaged bases and are also intermediates in base excision repair (43). Thymine glycols are generated by exposure to ionising and ultraviolet (UV) radiation and reactive oxygen species (44,45). The presence of an abasic site strongly inhibits Tgo-Pol and with a running start primer, noticeable stalled bands at +8 (immediately before the lesion) and +9 (flush with the lesion) (Figure 4A) were produced. This result suggests that the wild-type enzyme has difficulties both inserting a deoxynucleotide across an abasic site and also extending the mismatch produced. However, some read-through is apparent and full-length products are slowly formed. Single deoxynucleotide incorporation, using a primer designed to initiate synthesis immediately prior to the lesion (Figure 2A), shows the wild type prefers to insert purines opposite abasic sites, although pyrimidines can also be incorporated (Figure 4A). Tgo-Pol Z1 is more easily able to replicate through abasic sites; with the running start primer bands at +8 and +9 are apparent, but are less pronounced than for the wild type, and full extension occurs more rapidly. Tgo-Pol Z1 is capable of inserting all four bases opposite the lesion, with little discrimination between purines and pyrimidines (Figure 4A). Tgo-Pol Z1 was better than wild type at continuing synthesis from an abasic site:dA mis-pair, the expected product following initial insertion of dAMP opposite the basic site (Figure 4A). Both enzymes handled the thymine glycol in a near equivalent fashion and this lesion provides much less of a barrier, especially to the wild type, than does the abasic site (Figure 4B). With both the parent and Tgo-Pol Z1, evidence of stalling at +8 and +9 is apparent; nevertheless, full-length product builds up reasonably rapidly. Both polymerases pair the thymine glycol with adenine alone, insertion of the other three bases not taking place, and are able to extend reasonably efficiently from this mis-pair (Figure 4B).


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 primer-templates that contain a single damaged base in the template. (A) Copying of a template containing an abasic site (Ab) (Figure 2A; X = T, Y = abasic site) by Tgo-Pol and Tgo-Pol Z1. (A1) shows the results found with the running start primer and all four dNTPs. (A2) uses the primer that initiates polymerisation from just before the abasic site (Figure 2A) and only a single dNTP to determine preferential incorporation opposite the lesion. (A3) utilises a primer that has a dA base across from the abasic site (Figure 2A), enabling extension from the dA:abasic site mis-pair to be evaluated. (B) Copying of a template containing a thymine glycol (Thgl) (Figure 2A; X = T, Y = thymine glycol) by Tgo-Pol and Tgo-Pol Z1. (B1), (B2) and (B3) are identical to A1, A2 and A3, except a thymine glycol replaces the abasic site.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: Extension of primer-templates that contain a single damaged base in the template. (A) Copying of a template containing an abasic site (Ab) (Figure 2A; X = T, Y = abasic site) by Tgo-Pol and Tgo-Pol Z1. (A1) shows the results found with the running start primer and all four dNTPs. (A2) uses the primer that initiates polymerisation from just before the abasic site (Figure 2A) and only a single dNTP to determine preferential incorporation opposite the lesion. (A3) utilises a primer that has a dA base across from the abasic site (Figure 2A), enabling extension from the dA:abasic site mis-pair to be evaluated. (B) Copying of a template containing a thymine glycol (Thgl) (Figure 2A; X = T, Y = thymine glycol) by Tgo-Pol and Tgo-Pol Z1. (B1), (B2) and (B3) are identical to A1, A2 and A3, except a thymine glycol replaces the abasic site.
Mentions: To investigate the effects of introducing a damaged base into DNA, two types of lesions, an abasic site and a thymine glycol, were selected. Abasic sites can arise spontaneously by depurination/depyrimidation of both standard and damaged bases and are also intermediates in base excision repair (43). Thymine glycols are generated by exposure to ionising and ultraviolet (UV) radiation and reactive oxygen species (44,45). The presence of an abasic site strongly inhibits Tgo-Pol and with a running start primer, noticeable stalled bands at +8 (immediately before the lesion) and +9 (flush with the lesion) (Figure 4A) were produced. This result suggests that the wild-type enzyme has difficulties both inserting a deoxynucleotide across an abasic site and also extending the mismatch produced. However, some read-through is apparent and full-length products are slowly formed. Single deoxynucleotide incorporation, using a primer designed to initiate synthesis immediately prior to the lesion (Figure 2A), shows the wild type prefers to insert purines opposite abasic sites, although pyrimidines can also be incorporated (Figure 4A). Tgo-Pol Z1 is more easily able to replicate through abasic sites; with the running start primer bands at +8 and +9 are apparent, but are less pronounced than for the wild type, and full extension occurs more rapidly. Tgo-Pol Z1 is capable of inserting all four bases opposite the lesion, with little discrimination between purines and pyrimidines (Figure 4A). Tgo-Pol Z1 was better than wild type at continuing synthesis from an abasic site:dA mis-pair, the expected product following initial insertion of dAMP opposite the basic site (Figure 4A). Both enzymes handled the thymine glycol in a near equivalent fashion and this lesion provides much less of a barrier, especially to the wild type, than does the abasic site (Figure 4B). With both the parent and Tgo-Pol Z1, evidence of stalling at +8 and +9 is apparent; nevertheless, full-length product builds up reasonably rapidly. Both polymerases pair the thymine glycol with adenine alone, insertion of the other three bases not taking place, and are able to extend reasonably efficiently from this mis-pair (Figure 4B).

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