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Human PrimPol mutation associated with high myopia has a DNA replication defect.

Keen BA, Bailey LJ, Jozwiakowski SK, Doherty AJ - Nucleic Acids Res. (2014)

Bottom Line: Here, we examined whether this mutation resulted in any changes in the molecular and cellular activities associated with human PrimPol.We also demonstrate that the decreased activity of PrimPolY89D is associated with reduced affinities for DNA and nucleotides, resulting in diminished catalytic efficiency.This mutation also reduces cell viability after DNA damage and significantly slows replication fork rates in vivo.

View Article: PubMed Central - PubMed

Affiliation: Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton BN1 9RQ, UK.

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Related in: MedlinePlus

Processivity of human PrimPol and PrimPolY89D. Primer extension assays were performed in the presence of an excess of herring sperm DNA trap to ensure the polymerase only binds once to the template DNA. Control reactions were also carried out to test the effectiveness of the trap DNA (data not shown). The first lanes represent a no-enzyme control and lanes 2–6 represent time points of 15, 30, 60, 120 and 360 s. (A) Human PrimPol is a poorly processive enzyme, inserting up to four nucleotides opposite a templating DNA strand in a single binding event. (B) PrimPolY89D has marked reduction in processivity, and is only able to insert a single nucleotide opposite a templating DNA strand per single binding event. (C) PrimPolY89S can also insert only one single nucleotide opposite a templating DNA strand. (D) PrimPolY89F restores processivity and has the ability to insert up to four nucleotides, similarly to wild-type PrimPol.
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Figure 4: Processivity of human PrimPol and PrimPolY89D. Primer extension assays were performed in the presence of an excess of herring sperm DNA trap to ensure the polymerase only binds once to the template DNA. Control reactions were also carried out to test the effectiveness of the trap DNA (data not shown). The first lanes represent a no-enzyme control and lanes 2–6 represent time points of 15, 30, 60, 120 and 360 s. (A) Human PrimPol is a poorly processive enzyme, inserting up to four nucleotides opposite a templating DNA strand in a single binding event. (B) PrimPolY89D has marked reduction in processivity, and is only able to insert a single nucleotide opposite a templating DNA strand per single binding event. (C) PrimPolY89S can also insert only one single nucleotide opposite a templating DNA strand. (D) PrimPolY89F restores processivity and has the ability to insert up to four nucleotides, similarly to wild-type PrimPol.

Mentions: PrimPol was previously shown to be a distributive DNA polymerase, inserting only up to four nucleotides during a single binding event (Figure 4). We next determined whether the processivity of the mutant was altered, causing the observed reduction in the polymerase activity. PrimPolY89D variant could only insert a single nucleotide opposite a templating strand of DNA, making it an even more distributive enzyme than wild-type PrimPol (Figure 4). To determine whether the aromatic ring of tyrosine was the main determinant for enzyme activity, we tested the processivity activities of Y89S and Y89F variants. Consistent with Y89D, PrimPolY89S could only insert a single nucleotide. However, in common with the wild-type enzyme, PrimPolY89F could insert up to four nucleotides. This establishes that the presence of an aromatic side-chain at this position is requisite for maintaining more processive extension by PrimPol.


Human PrimPol mutation associated with high myopia has a DNA replication defect.

Keen BA, Bailey LJ, Jozwiakowski SK, Doherty AJ - Nucleic Acids Res. (2014)

Processivity of human PrimPol and PrimPolY89D. Primer extension assays were performed in the presence of an excess of herring sperm DNA trap to ensure the polymerase only binds once to the template DNA. Control reactions were also carried out to test the effectiveness of the trap DNA (data not shown). The first lanes represent a no-enzyme control and lanes 2–6 represent time points of 15, 30, 60, 120 and 360 s. (A) Human PrimPol is a poorly processive enzyme, inserting up to four nucleotides opposite a templating DNA strand in a single binding event. (B) PrimPolY89D has marked reduction in processivity, and is only able to insert a single nucleotide opposite a templating DNA strand per single binding event. (C) PrimPolY89S can also insert only one single nucleotide opposite a templating DNA strand. (D) PrimPolY89F restores processivity and has the ability to insert up to four nucleotides, similarly to wild-type PrimPol.
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Figure 4: Processivity of human PrimPol and PrimPolY89D. Primer extension assays were performed in the presence of an excess of herring sperm DNA trap to ensure the polymerase only binds once to the template DNA. Control reactions were also carried out to test the effectiveness of the trap DNA (data not shown). The first lanes represent a no-enzyme control and lanes 2–6 represent time points of 15, 30, 60, 120 and 360 s. (A) Human PrimPol is a poorly processive enzyme, inserting up to four nucleotides opposite a templating DNA strand in a single binding event. (B) PrimPolY89D has marked reduction in processivity, and is only able to insert a single nucleotide opposite a templating DNA strand per single binding event. (C) PrimPolY89S can also insert only one single nucleotide opposite a templating DNA strand. (D) PrimPolY89F restores processivity and has the ability to insert up to four nucleotides, similarly to wild-type PrimPol.
Mentions: PrimPol was previously shown to be a distributive DNA polymerase, inserting only up to four nucleotides during a single binding event (Figure 4). We next determined whether the processivity of the mutant was altered, causing the observed reduction in the polymerase activity. PrimPolY89D variant could only insert a single nucleotide opposite a templating strand of DNA, making it an even more distributive enzyme than wild-type PrimPol (Figure 4). To determine whether the aromatic ring of tyrosine was the main determinant for enzyme activity, we tested the processivity activities of Y89S and Y89F variants. Consistent with Y89D, PrimPolY89S could only insert a single nucleotide. However, in common with the wild-type enzyme, PrimPolY89F could insert up to four nucleotides. This establishes that the presence of an aromatic side-chain at this position is requisite for maintaining more processive extension by PrimPol.

Bottom Line: Here, we examined whether this mutation resulted in any changes in the molecular and cellular activities associated with human PrimPol.We also demonstrate that the decreased activity of PrimPolY89D is associated with reduced affinities for DNA and nucleotides, resulting in diminished catalytic efficiency.This mutation also reduces cell viability after DNA damage and significantly slows replication fork rates in vivo.

View Article: PubMed Central - PubMed

Affiliation: Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton BN1 9RQ, UK.

Show MeSH
Related in: MedlinePlus