Human PrimPol mutation associated with high myopia has a DNA replication defect.
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.
Affiliation: Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton BN1 9RQ, UK.Show MeSH
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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.
Affiliation: Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton BN1 9RQ, UK.