6608–6619 Nucleic Acids Research, 2012, Vol. 40, No. 14 doi:10.1093/nar/gks318 Published online 15 April 2012 Impact of PNKP mutations associated with microcephaly, seizures and developmental delay on enzyme activity and DNA strand break repair John J. Reynolds 1 , Alexandra K. Walker1, Edward C. Gilmore2,3, Christopher A. Walsh2,4 and Keith W. Caldecott1,* 1Genome Damage and Stability Centre, University of Sussex, Science Park Road, Falmer, Brighton, BN1 9RQ, UK, 2Division of Genetics, Manton Centre for Orphan Disease, Howard Hughes Medical Institute, Children’s Hospital, Harvard Medical School, Boston, MA 02115, 3Division of Child Neurology, Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114 and 4Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA 02142, USA Received January 4, 2012; Revised March 26, 2012; Accepted March 27, 2012 ABSTRACT 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 50-kinase and DNA 30-phos- phatase 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 muta- tions likely ablating cellular DNA 50-kinase activity and all of the mutations greatly reducing cellular DNA 30-phosphatase activity. INTRODUCTION Discontinuities in one strand of the DNA double helix, denoted DNA single-strand breaks (SSBs), are the most commonly occurring type of DNA lesion in cells, with tens of thousands arising per cell per day (1,2). SSBs can arise either directly from the disintegration of damaged sugars or indirectly from abortive topoisomerase 1 activity or during the removal of damaged DNA bases or deoxyribose by DNA base excision repair. If not repaired quickly enough, SSBs can block transcription or result in the formation of DNA double-strand breaks (DSBs) during DNA replication (3,4). One source of SSBs is attack of DNA by reactive oxygen species (ROS) generated during normal cellular metabolism (5–7). To counter this threat, cells employ a number of DNA repair pathways collectively termed DNA single-strand break repair (SSBR) to ensure the rapid and efﬁcient removal of SSBs (8). SSBR typically involves four stages of repair: detection of the break, DNA end processing, DNA gap ﬁlling and DNA ligation. During DNA end processing, ‘damaged’ DNA termini are restored to conventional 30-hydroxyl and 50-phosphate moieties to enable subsequent steps of SSBR to occur. Enzymatically, this is the most diverse step because of the variety of types of damaged DNA termini that can arise. The importance of DNA end processing is indicated by the existence of neuropathological disorders in which proteins involved this process are mutated. These are ataxia oculomotor apraxia-1 (AOA1), spinocerebellar ataxia with axonal neuropathy-1 (SCAN1) and micro- cephaly with early-onset intractable seizures and develop- mental delay (MCSZ), which are caused by mutations in aprataxin (APTX), tyrosyl–DNA phosphodiesterase 1 (TDP1) and polynucleotide kinase/phosphatase (PNKP), respectively (9–12). A striking feature of these diseases is that they lack any major extraneurological symptoms, suggesting that the development and/or maintenance of neurones is hypersensitive to defects in DNA end *To whom correspondence should be addressed. Tel: +44 1273 877519; Fax: +44 1273 678121; Email: k.w.caldecott@sussex.ac.uk The Author(s) 2012. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.