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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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Oligodeoxynucleotides used in this study. (A) The 50 base template (written in 3′→5′ direction) containing various damaged bases (X = T, Y = T (control); X = T, Y = abasic site; X = T, Y = thymine glycol; X/Y = cis/syn thymine–thymine cyclobutane dimer) was hybridised with one of four primers (written in the 5′→3′ direction). The shortest primer allows running start read-through of the lesions. The longer primers allow extension to begin: (1) at the base immediately preceding the lesion; (2) directly opposite the abasic site or thymine glycol; (3) from either of the damaged bases in the thymine dimer. (B) and (C) Primer-templates designed to initiate DNA synthesis at mismatched C:T and T:T base pairs, respectively. (D) The substrates used to investigate the thymine 6–4 photoproduct dimer. The template strand (written in the 3′→5′ direction, ZZ represents the 6–4 photoproduct) was hybridised with one of four primers (written in the 5′→3′ direction). The primers allow synthesis to commence from: (1) the base directly before the lesion; (2) the 3′ base of the 6–4 photoproduct; (3) the 5′ base of the 6–4 photoproduct; (4) the base immediately following the lesion. All the primers shown in the figure were 5′-end labelled with hexachlorofluorescein (H).
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Figure 2: Oligodeoxynucleotides used in this study. (A) The 50 base template (written in 3′→5′ direction) containing various damaged bases (X = T, Y = T (control); X = T, Y = abasic site; X = T, Y = thymine glycol; X/Y = cis/syn thymine–thymine cyclobutane dimer) was hybridised with one of four primers (written in the 5′→3′ direction). The shortest primer allows running start read-through of the lesions. The longer primers allow extension to begin: (1) at the base immediately preceding the lesion; (2) directly opposite the abasic site or thymine glycol; (3) from either of the damaged bases in the thymine dimer. (B) and (C) Primer-templates designed to initiate DNA synthesis at mismatched C:T and T:T base pairs, respectively. (D) The substrates used to investigate the thymine 6–4 photoproduct dimer. The template strand (written in the 3′→5′ direction, ZZ represents the 6–4 photoproduct) was hybridised with one of four primers (written in the 5′→3′ direction). The primers allow synthesis to commence from: (1) the base directly before the lesion; (2) the 3′ base of the 6–4 photoproduct; (3) the 5′ base of the 6–4 photoproduct; (4) the base immediately following the lesion. All the primers shown in the figure were 5′-end labelled with hexachlorofluorescein (H).

Mentions: The primer-templates used in these studies are illustrated in Figure 2. Most are based on templates 50 bases in length that contain a damaged base (abasic site, thymine glycol or cis-syn thymine–thymine cyclobutane pyrimidine dimer) 22 bases from the 5′ end (22 and 23 in case of the dimer). A number of primers, all labelled at their 5′-ends with hexachlorofluorescein for detection purposes, were applied. These could position the 3′ base used to initiate extension far upstream of the template modification (allowing running start read-through) or immediately in front of the lesion (allowing single base addition opposite the damaged base). Alternatively, the primer could terminate flush with the lesion, permitting study of extension from the resulting canonical base: damaged base mismatches (Figure 2A). Two primer-templates lack modified bases, designed, instead, to allow polymerisation to begin from a C:T or a T:T mismatch at 3′ end of the primer (Figure 2B and C). Due to the difficulties with synthesizing the thymine 6–4 photoproduct dimer, this lesion was present in a 30 base template, 13 and 14 bases from the 5′ end. Several primers that terminated just prior to the lesion, at the 3′ and 5′ bases of the lesion and immediately post lesion, were employed (Figure 2D).


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)

Oligodeoxynucleotides used in this study. (A) The 50 base template (written in 3′→5′ direction) containing various damaged bases (X = T, Y = T (control); X = T, Y = abasic site; X = T, Y = thymine glycol; X/Y = cis/syn thymine–thymine cyclobutane dimer) was hybridised with one of four primers (written in the 5′→3′ direction). The shortest primer allows running start read-through of the lesions. The longer primers allow extension to begin: (1) at the base immediately preceding the lesion; (2) directly opposite the abasic site or thymine glycol; (3) from either of the damaged bases in the thymine dimer. (B) and (C) Primer-templates designed to initiate DNA synthesis at mismatched C:T and T:T base pairs, respectively. (D) The substrates used to investigate the thymine 6–4 photoproduct dimer. The template strand (written in the 3′→5′ direction, ZZ represents the 6–4 photoproduct) was hybridised with one of four primers (written in the 5′→3′ direction). The primers allow synthesis to commence from: (1) the base directly before the lesion; (2) the 3′ base of the 6–4 photoproduct; (3) the 5′ base of the 6–4 photoproduct; (4) the base immediately following the lesion. All the primers shown in the figure were 5′-end labelled with hexachlorofluorescein (H).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: Oligodeoxynucleotides used in this study. (A) The 50 base template (written in 3′→5′ direction) containing various damaged bases (X = T, Y = T (control); X = T, Y = abasic site; X = T, Y = thymine glycol; X/Y = cis/syn thymine–thymine cyclobutane dimer) was hybridised with one of four primers (written in the 5′→3′ direction). The shortest primer allows running start read-through of the lesions. The longer primers allow extension to begin: (1) at the base immediately preceding the lesion; (2) directly opposite the abasic site or thymine glycol; (3) from either of the damaged bases in the thymine dimer. (B) and (C) Primer-templates designed to initiate DNA synthesis at mismatched C:T and T:T base pairs, respectively. (D) The substrates used to investigate the thymine 6–4 photoproduct dimer. The template strand (written in the 3′→5′ direction, ZZ represents the 6–4 photoproduct) was hybridised with one of four primers (written in the 5′→3′ direction). The primers allow synthesis to commence from: (1) the base directly before the lesion; (2) the 3′ base of the 6–4 photoproduct; (3) the 5′ base of the 6–4 photoproduct; (4) the base immediately following the lesion. All the primers shown in the figure were 5′-end labelled with hexachlorofluorescein (H).
Mentions: The primer-templates used in these studies are illustrated in Figure 2. Most are based on templates 50 bases in length that contain a damaged base (abasic site, thymine glycol or cis-syn thymine–thymine cyclobutane pyrimidine dimer) 22 bases from the 5′ end (22 and 23 in case of the dimer). A number of primers, all labelled at their 5′-ends with hexachlorofluorescein for detection purposes, were applied. These could position the 3′ base used to initiate extension far upstream of the template modification (allowing running start read-through) or immediately in front of the lesion (allowing single base addition opposite the damaged base). Alternatively, the primer could terminate flush with the lesion, permitting study of extension from the resulting canonical base: damaged base mismatches (Figure 2A). Two primer-templates lack modified bases, designed, instead, to allow polymerisation to begin from a C:T or a T:T mismatch at 3′ end of the primer (Figure 2B and C). Due to the difficulties with synthesizing the thymine 6–4 photoproduct dimer, this lesion was present in a 30 base template, 13 and 14 bases from the 5′ end. Several primers that terminated just prior to the lesion, at the 3′ and 5′ bases of the lesion and immediately post lesion, were employed (Figure 2D).

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