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Nucl._Acids_Res.-2012-Reynolds-6608-19.pdf (7.14 MB)

Impact of PNKP mutations associated with microcephaly, seizures and developmental delay on enzyme activity and DNA strand break repair

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posted on 2023-06-08, 11:22 authored by John J Reynolds, Alexandra K Walker, Edward C Gilmore, Christopher A Walsh, Keith CaldecottKeith Caldecott
Microcephaly with early-onset, intractable seizures and developmental delay (MCSZ) is a hereditary disease caused by mutations in polynucleotide kinase/phosphatase (PNKP), a DNA strand break repair protein with DNA 5'-kinase and DNA 3'-phosphatase activity. To investigate the molecular basis of this disease, we examined the impact of MCSZ mutations on PNKP activity in vitro and in cells. Three of the four mutations currently associated with MCSZ greatly reduce or ablate DNA kinase activity of recombinant PNKP at 30°C (L176F, T424Gfs48X and exon15?fs4X), but only one of these mutations reduces DNA phosphatase activity under the same conditions (L176F). The fourth mutation (E326K) has little impact on either DNA kinase or DNA phosphatase activity at 30°C, but is less stable than the wild-type enzyme at physiological temperature. Critically, all of the MCSZ mutations identified to date result in ~10-fold reduced cellular levels of PNKP protein, and reduced rates of chromosomal DNA strand break repair. Together, these data suggest that all four known MCSZ mutations reduce the cellular stability and level of PNKP protein, with three mutations likely ablating cellular DNA 5'-kinase activity and all of the mutations greatly reducing cellular DNA 3'-phosphatase activity.

History

Publication status

  • Published

File Version

  • Published version

Journal

Nucleic Acids Research

ISSN

1362-4962

Publisher

Oxford University Press

Issue

14

Volume

40

Page range

6608-6619

Department affiliated with

  • Sussex Centre for Genome Damage Stability Publications

Full text available

  • Yes

Peer reviewed?

  • Yes

Legacy Posted Date

2012-05-09

First Open Access (FOA) Date

2016-03-22

First Compliant Deposit (FCD) Date

2016-11-10

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