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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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PrimPol Y89D cannot fully complement the UV sensitivity seen in PrimPol−/− DT40 cells due to decreased replication fork speeds. (A) Western blot showing overexpression of wild type and Y89D point mutant forms of PrimPol in PrimPol knockout (PrimPol−/−) DT40 chicken cells. (B) Cell viability was measured using CellTiter-Blue 48 h after increasing doses of UV-C were administered to the different cell types. Results represent three independent repeats and the error bars represent the standard deviation across the repeats. (C) DNA fibres were measured after sequential labelling with CldU and IdU for 20 min each to calculate replication speed. Results represent three independent repeats. (D) A 20 J/m2 UV-C pulse was included between the two labels and the ratio of the pre- and post-UV-C labels was calculated, with increased ratios representing replication stalling after UV. Results represent three independent repeats.
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Figure 8: PrimPol Y89D cannot fully complement the UV sensitivity seen in PrimPol−/− DT40 cells due to decreased replication fork speeds. (A) Western blot showing overexpression of wild type and Y89D point mutant forms of PrimPol in PrimPol knockout (PrimPol−/−) DT40 chicken cells. (B) Cell viability was measured using CellTiter-Blue 48 h after increasing doses of UV-C were administered to the different cell types. Results represent three independent repeats and the error bars represent the standard deviation across the repeats. (C) DNA fibres were measured after sequential labelling with CldU and IdU for 20 min each to calculate replication speed. Results represent three independent repeats. (D) A 20 J/m2 UV-C pulse was included between the two labels and the ratio of the pre- and post-UV-C labels was calculated, with increased ratios representing replication stalling after UV. Results represent three independent repeats.

Mentions: 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. We utilized a DT40 PrimPol deleted cell line (PrimPol −/−) (4) to examine the ability of PrimPolY89D to complement this loss, as previously reported for other mutant forms of PrimPol (14). We stably expressed human PrimPolY89D at similar levels to WT PrimPol in PrimPol knockout cells (Figure 8A), with no obvious effects on cell growth.


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

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

PrimPol Y89D cannot fully complement the UV sensitivity seen in PrimPol−/− DT40 cells due to decreased replication fork speeds. (A) Western blot showing overexpression of wild type and Y89D point mutant forms of PrimPol in PrimPol knockout (PrimPol−/−) DT40 chicken cells. (B) Cell viability was measured using CellTiter-Blue 48 h after increasing doses of UV-C were administered to the different cell types. Results represent three independent repeats and the error bars represent the standard deviation across the repeats. (C) DNA fibres were measured after sequential labelling with CldU and IdU for 20 min each to calculate replication speed. Results represent three independent repeats. (D) A 20 J/m2 UV-C pulse was included between the two labels and the ratio of the pre- and post-UV-C labels was calculated, with increased ratios representing replication stalling after UV. Results represent three independent repeats.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231748&req=5

Figure 8: PrimPol Y89D cannot fully complement the UV sensitivity seen in PrimPol−/− DT40 cells due to decreased replication fork speeds. (A) Western blot showing overexpression of wild type and Y89D point mutant forms of PrimPol in PrimPol knockout (PrimPol−/−) DT40 chicken cells. (B) Cell viability was measured using CellTiter-Blue 48 h after increasing doses of UV-C were administered to the different cell types. Results represent three independent repeats and the error bars represent the standard deviation across the repeats. (C) DNA fibres were measured after sequential labelling with CldU and IdU for 20 min each to calculate replication speed. Results represent three independent repeats. (D) A 20 J/m2 UV-C pulse was included between the two labels and the ratio of the pre- and post-UV-C labels was calculated, with increased ratios representing replication stalling after UV. Results represent three independent repeats.
Mentions: 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. We utilized a DT40 PrimPol deleted cell line (PrimPol −/−) (4) to examine the ability of PrimPolY89D to complement this loss, as previously reported for other mutant forms of PrimPol (14). We stably expressed human PrimPolY89D at similar levels to WT PrimPol in PrimPol knockout cells (Figure 8A), with no obvious effects on cell growth.

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