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Nucleic Acids Res DOI:10.1093/nar/gks318

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

Publication TypeJournal Article
Year of Publication2012
AuthorsReynolds, JJ, Walker, AK, Gilmore, EC, Walsh, CA, Caldecott, KW
JournalNucleic Acids Res
Date Published2012 Aug
KeywordsCell Line, Developmental Disabilities, DNA Breaks, Single-Stranded, DNA Repair, DNA Repair Enzymes, Enzyme Stability, Humans, Microcephaly, Mutation, Phosphotransferases (Alcohol Group Acceptor), Seizures

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 exon15Δfs4X), 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.


Alternate JournalNucleic Acids Res.
PubMed ID22508754
PubMed Central IDPMC3413127
Grant ListMR/J006750/1 / / Medical Research Council / United Kingdom
R01 NS035129 / NS / NINDS NIH HHS / United States
C6563/A10192 / / Cancer Research UK / United Kingdom
G0600776 / / Medical Research Council / United Kingdom