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: To evaluate the effects of this mutation on the enzymatic properties of PrimPol, a kinetic analysis was performed to determine the catalytic efficiency (kpol) and binding constants for dATP (KD(dNTP)) opposite a templating thymine (Supplementary Figure S1). We found the wild-type kpol to be 6.98 min−1 and the KD(dNTP) to be 15.51 μM (Figure 6). PrimPol is an extremely slow enzyme—for comparison the kpol of base excision repair enzyme DNA polymerase β is 1944 min−1 (17). We determined the Y89D variant of PrimPol to be a slightly slower polymerase, with a kpol of 4.07 min−1 and binds dNTPs over 10-fold less competently, with a KD(dNTP) of 170.21 μM (Figure 6). The reduction in catalytic efficiency and dramatic reduction in dNTP binding may or may not be dependent on the initial binding of DNA.
Affiliation: Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton BN1 9RQ, UK.