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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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Kinetic analysis of human PrimPol and PrimPolY89D. Single turnover kinetics of wild-type and PrimPolY89D to determine catalytic efficiency (kpol) and dNTP binding efficiency (KD(dNTP)). (A) The concentration of extended DNA product, as determined by electrophoresis, was plotted against time and fit to an exponential curve for a number of dATP concentrations for wild-type and PrimPolY89D. These data were fit to exponential curves as described in Equation (1) and kobs was determined. (B) kobs was subsequently plot against dATP concentration for wild-type PrimPol and PrimPolY89D and these data were fit to hyperbolic curves as described in Equation (2) to determine kpol and KD(dNTP). kpol for wild-type PrimPol was found to be 6.98 min−1 ± 0.40 and KD(dNTP) was 15.51 μM ± 2.71. kpol for PrimPolY89D was 4.07 min−1 ± 0.19 and KD(dNTP) was 170.21 μM ± 18.80.
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Figure 6: Kinetic analysis of human PrimPol and PrimPolY89D. Single turnover kinetics of wild-type and PrimPolY89D to determine catalytic efficiency (kpol) and dNTP binding efficiency (KD(dNTP)). (A) The concentration of extended DNA product, as determined by electrophoresis, was plotted against time and fit to an exponential curve for a number of dATP concentrations for wild-type and PrimPolY89D. These data were fit to exponential curves as described in Equation (1) and kobs was determined. (B) kobs was subsequently plot against dATP concentration for wild-type PrimPol and PrimPolY89D and these data were fit to hyperbolic curves as described in Equation (2) to determine kpol and KD(dNTP). kpol for wild-type PrimPol was found to be 6.98 min−1 ± 0.40 and KD(dNTP) was 15.51 μM ± 2.71. kpol for PrimPolY89D was 4.07 min−1 ± 0.19 and KD(dNTP) was 170.21 μM ± 18.80.

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.


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

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

Kinetic analysis of human PrimPol and PrimPolY89D. Single turnover kinetics of wild-type and PrimPolY89D to determine catalytic efficiency (kpol) and dNTP binding efficiency (KD(dNTP)). (A) The concentration of extended DNA product, as determined by electrophoresis, was plotted against time and fit to an exponential curve for a number of dATP concentrations for wild-type and PrimPolY89D. These data were fit to exponential curves as described in Equation (1) and kobs was determined. (B) kobs was subsequently plot against dATP concentration for wild-type PrimPol and PrimPolY89D and these data were fit to hyperbolic curves as described in Equation (2) to determine kpol and KD(dNTP). kpol for wild-type PrimPol was found to be 6.98 min−1 ± 0.40 and KD(dNTP) was 15.51 μM ± 2.71. kpol for PrimPolY89D was 4.07 min−1 ± 0.19 and KD(dNTP) was 170.21 μM ± 18.80.
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Figure 6: Kinetic analysis of human PrimPol and PrimPolY89D. Single turnover kinetics of wild-type and PrimPolY89D to determine catalytic efficiency (kpol) and dNTP binding efficiency (KD(dNTP)). (A) The concentration of extended DNA product, as determined by electrophoresis, was plotted against time and fit to an exponential curve for a number of dATP concentrations for wild-type and PrimPolY89D. These data were fit to exponential curves as described in Equation (1) and kobs was determined. (B) kobs was subsequently plot against dATP concentration for wild-type PrimPol and PrimPolY89D and these data were fit to hyperbolic curves as described in Equation (2) to determine kpol and KD(dNTP). kpol for wild-type PrimPol was found to be 6.98 min−1 ± 0.40 and KD(dNTP) was 15.51 μM ± 2.71. kpol for PrimPolY89D was 4.07 min−1 ± 0.19 and KD(dNTP) was 170.21 μM ± 18.80.
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.

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