Polynucleotide kinase-phosphatase enables neurogenesis via multiple DNA repair pathways to maintain genome stability

Polynucleotide kinase–phosphatase (PNKP) is a DNA repair factor possessing both 5′‐kinase and 3′‐phosphatase activities to modify ends of a DNA break prior to ligation. Recently, decreased PNKP levels were identified as the cause of severe neuropathology present in the human microcephaly with seizures (MCSZ) syndrome. Utilizing novel murine Pnkp alleles that attenuate expression and a T424GfsX48 frame‐shift allele identified in MCSZ individuals, we determined how PNKP inactivation impacts neurogenesis. Mice with PNKP inactivation in neural progenitors manifest neurodevelopmental abnormalities and postnatal death. This severe phenotype involved defective base excision repair and non‐homologous end‐joining, pathways required for repair of both DNA single‐ and double‐strand breaks. Although mice homozygous for the T424GfsX48 allele were lethal embryonically, attenuated PNKP levels (akin to MCSZ) showed general neurodevelopmental defects, including microcephaly, indicating a critical developmental PNKP threshold. Directed postnatal neural inactivation of PNKP affected specific subpopulations including oligodendrocytes, indicating a broad requirement for genome maintenance, both during and after neurogenesis. These data illuminate the basis for selective neural vulnerability in DNA repair deficiency disease. Synopsis Polynucleotide kinase‐phosphatase (PNKP), a repair factor that modifies DNA breaks prior to ligation, is essential for normal brain development by preventing the accumulation of endogenous DNA breaks, which otherwise results in widespread cell death during neurogenesis. PNKP is essential for neurogenesis by preventing DNA damage accumulation. PNKP is required for efficient base‐excision repair (BER) of single‐strand breaks as well as non‐homologous end joining (NHEJ) repair of double‐strand breaks. Attenuated PNKP levels generated using a hypomorphic Pnkp allele compromised brain development and generate a microcephaly with seizures (MCSZ)‐like neural phenotype. Postnatal inactivation of PNKP in various neural compartments resulted in defective cellular homeostasis in mature cell types in the brain, including select neurons and oligodendrocytes. Graphical Abstract Polynucleotide kinase‐phosphatase is essential for normal brain development by preventing the accumulation of endogenous DNA single‐ and double‐strand breaks, which otherwise results in widespread cell death during neurogenesis.