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

Benjamin A Keen,Stanislaw K Jozwiakowski,Aidan J Doherty,Laura J Bailey

doi:10.1093/nar/gku879

Benjamin A Keen, Stanislaw K Jozwiakowski + Show 2 more

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https://doi.org/10.1093/nar/gku879

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Journal: Nucleic acids research	Publication Date: Sep 27, 2014
Citations: 41	License type: CC BY 4.0

Affiliation: University of Sussex

Abstract

PrimPol is a primase-polymerase found in humans, and other eukaryotes, involved in bypassing lesions encountered during DNA replication. PrimPol employs both translesion synthesis and repriming mechanisms to facilitate lesion bypass by the replisome. PrimPol has been reported to be a potential susceptibility gene associated with the development of myopia. Mutation of tyrosine 89 to aspartic acid (PrimPolY89D) has been identified in a number of cases of high myopia, implicating it in the aetiology of this disorder. Here, we examined whether this mutation resulted in any changes in the molecular and cellular activities associated with human PrimPol. We show that PrimPolY89D has a striking decrease in primase and polymerase activities. The hydrophobic ring of tyrosine is important for retaining wild-type extension activity. We also demonstrate that the decreased activity of PrimPolY89D is associated with reduced affinities for DNA and nucleotides, resulting in diminished catalytic efficiency. Although the structure and stability of PrimPolY89D is altered, its fidelity remains unchanged. This mutation also reduces cell viability after DNA damage and significantly slows replication fork rates in vivo. Together, these findings establish that the major DNA replication defect associated with this PrimPol mutant is likely to contribute to the onset of high myopia.

Highlights

Cells are required to replicate their genomes in a faithful way to avoid mutagenesis maintaining genetic stability
To determine if PrimPol displays an underlying biochemical defect that contributes to the high myopia clinical phenotype, we first examined the primase activity of the PrimPol Y89D (PrimPolY89D) variant
To gain further insights into how the Y89D mutant form of PrimPol may contribute to the myopic phenotype observed in human patients, we studied the influence of PrimPolY89D on DNA replication in damaged and undamaged cells

Summary

Introduction

Cells are required to replicate their genomes in a faithful way to avoid mutagenesis maintaining genetic stability. One of the many undesirable consequences of such damage is the disruption of the progression of DNA replicases [1], which copy the genetic material These replication machines are exquisitely sensitive to the conformation of the template and are prone to stalling upon encountering DNA damage. To overcome such genetic obstacles, cells have evolved a variety of lesion tolerance pathways to allow bypass of damage and resumption of replication [2,3]. PrimPol is required for the replication past particular lesions, such as UV photoproducts, that block the cellular replication machinery It can deploy two different mechanisms, utilizing either of its polymerase and primase activities to facilitate efficient lesion bypass. PrimPol knockout cells are sensitive to damaging agents (e.g. UV, 4NQO and MMS) and murine PrimPol−/− cells show significant levels of DNA breaks, after treatment with agents that stall replication [4]

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