Mutations to Ku reveal differences in human somatic cell lines

Fattah, Kazi R.; Ruis, Brian L.; Hendrickson, Eric A.

doi:10.1016/j.dnarep.2008.02.008

Cited by 39 publications

(47 citation statements)

References 52 publications

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“…28,29). Similarly, targeting efficiency, which is mediated by HR, is increased in a dose-dependent manner (51). We have suggested that differing requirements for DNA-PK levels in humans may reflect a more stringent requirement to regulate HR at hyper-recombinogenic genomic regions (30,52).…”

Section: Figurementioning

confidence: 89%

PRKDC mutations in a SCID patient with profound neurological abnormalities

Woodbine¹,

Neal²,

Sasi³

et al. 2013

J. Clin. Invest.

134

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The DNA-dependent protein kinase catalytic subunit (DNA-PKcs; encoded by PRKDC) functions in DNA non-homologous end-joining (NHEJ), the major DNA double strand break (DSB) rejoining pathway. NHEJ also functions during lymphocyte development, joining V(D)J recombination intermediates during antigen receptor gene assembly. Here, we describe a patient with compound heterozygous mutations in PRKDC, low DNA-PKcs expression, barely detectable DNA-PK kinase activity, and impaired DSB repair. In a heterologous expression system, we found that one of the PRKDC mutations inactivated DNA-PKcs, while the other resulted in dramatically diminished but detectable residual function. The patient suffered SCID with reduced or absent T and B cells, as predicted from PRKDC-deficient animal models. Unexpectedly, the patient was also dysmorphic; showed severe growth failure, microcephaly, and seizures; and had profound, globally impaired neurological function. MRI scans revealed microcephaly-associated cortical and hippocampal dysplasia and progressive atrophy over 2 years of life. These neurological features were markedly more severe than those observed in patients with deficiencies in other NHEJ proteins. Although loss of DNA-PKcs in mice, dogs, and horses was previously shown not to impair neuronal development, our findings demonstrate a stringent requirement for DNA-PKcs during human neuronal development and suggest that high DNA-PK protein expression is required to sustain efficient pre-and postnatal neurogenesis. Introduction DNA nonhomologous end-joining (NHEJ) represents the major DNA double strand break (DSB) repair process in mammalian cells (1, 2). Thus, cells, mice, and patients defective in NHEJ proteins show markedly reduced DSB repair and radiosensitivity. NHEJ also functions during immune development due to its requisite role in V(D)J recombination (3). V(D)J recombination mediates immunoglobulin and T cell receptor gene assembly from variable (V), diversity (D), and joining (J) gene segments. Recombination is initiated by the RAG1/2 endonuclease, which introduces DSBs at recombination signal sequences (RSSs). The DNA ends, including the sequences encoding the antigen receptors (termed coding ends), have hairpinned termini; the RSSs are blunt ended. Rejoining of these recombination intermediates requires NHEJ proteins. Consequently, NHEJ defects in animals and humans causes SCID with reduced or absent T and B cells.6 core NHEJ components assemble as 2 complexes (1). The Ku70/80 heterodimer is the DNA double-stranded (ds) end recognition protein, binding DNA ds ends with high affinity. Ku recruits the DNA-dependent protein kinase catalytic subunit (DNA-PKcs; encoded by PRKDC), generating the DNA-PK holoenzyme. The DNA-PK complex prevents unbridled exonuclease digestion of DNA ends, enhances appropriate end-processing, and recruits the ligation complex, which encompasses XRCC4,

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Section: Figurementioning

confidence: 89%

PRKDC mutations in a SCID patient with profound neurological abnormalities

Woodbine¹,

Neal²,

Sasi³

et al. 2013

J. Clin. Invest.

134

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show abstract

“…In contrast, human cell lines with inactivated KU70 or KU80 are not viable, which suggests that KU70 and KU80 are essential for human cell viability (53,54). Xrcc4-deficient mice are embryonic lethal due to apoptosis of postmitotic neurons (55), and if this can be extrapolated to humans, functional null mutations in XRCC4 are also unlikely.…”

Section: Ku70- and Ku80-knockout Mice Are Viable And Have An Rs-scidmentioning

confidence: 99%

A DNA-PKcs mutation in a radiosensitive T–B– SCID patient inhibits Artemis activation and nonhomologous end-joining

et al. 2008

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Radiosensitive T -B -severe combined immunodeficiency (RS-SCID) is caused by defects in the nonhomologous end-joining (NHEJ) DNA repair pathway, which results in failure of functional V(D)J recombination. Here we have identified the first human RS-SCID patient to our knowledge with a DNA-PKcs missense mutation (L3062R). The causative mutation did not affect the kinase activity or DNA end-binding capacity of DNA-PKcs itself; rather, the presence of long P-nucleotide stretches in the immunoglobulin coding joints indicated that it caused insufficient Artemis activation, something that is dependent on Artemis interaction with autophosphorylated DNA-PKcs. Moreover, overall end-joining activity was hampered, suggesting that Artemis-independent DNA-PKcs functions were also inhibited. This study demonstrates that the presence of DNA-PKcs kinase activity is not sufficient to rule out a defect in this gene during diagnosis and treatment of RS-SCID patients. Further, the data suggest that residual DNA-PKcs activity is indispensable in humans.Introduction SCID is an inherited primary immunodeficiency. SCID patients present in the first year of life with severe opportunistic infections, chronic diarrhea, and failure to thrive. The total group of SCID patients can be divided in 2 main categories: those with T -B + SCID, who have a T cell signaling defect (70%), and those with T -B -SCID, who have a defect in V(D)J recombination (30%). V(D)J recombination assembles variable (V), diversity (D), and joining (J) gene segments of the Ig and TCR genes during B and T cell differentiation in order to generate a broad repertoire of antigen-specific receptors. V(D)J recombination starts with introduction of DNA breaks at the border of the gene segments and the flanking recombination signal sequences (RSSs) by the RAG1 and RAG2 proteins (1). The resulting blunt signal ends are ligated directly, forming a signal joint. The hairpin sealed coding ends require further processing before coding joint formation can occur. Recognition and repair of the DNA ends occur via the general nonhomologous end-joining (NHEJ) pathway of DNA double-strand break (DSB) repair (2, 3).DSBs induce ATM kinase activity, which phosphorylates histone H2AX (4), followed by binding of 53BP1, MDC1, and a complex of MRE11, RAD50, and NBS1 (MRN complex) (5, 6). These proteins form a microenvironment that holds together the DNA ends over a relatively large distance but still allows some degree of freedom for movement of the DNA ends and access of NHEJ proteins (7).

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“…The structural components of the core NHEJ complex, KU70 and KU80, are essential for human cell viability [24,25] and there are no documented patients with mutations in these proteins. Other components of the NHEJ complex (DNA-PKcs, Lig4 and XLF/Cernunnos) are also likely to be essential for development, although patients carrying hypomorphic mutations in these proteins have been reported [26][27][28].…”

Section: Introductionmentioning

confidence: 99%

DNA strand break repair and neurodegeneration

Rulten

Caldecott

2013

DNA Repair

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A number of DNA repair disorders are known to cause neurological problems. These disorders can be broadly characterised into early developmental, mid-to-late developmental or progressive. The exact developmental processes that are affected can influence disease pathology, with symptoms ranging from early embryonic lethality to late-onset ataxia. The category these diseases belong to depends on the frequency of lesions arising in the brain, the role of the defective repair pathway, and the nature of the mutation within the patient. Using observations from patients and transgenic mice, we discuss the importance of double strand break repair during neuroprogenitor proliferation and brain development and the repair of single stranded lesions in neuronal function and maintenance.

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Mutations to Ku reveal differences in human somatic cell lines

Cited by 39 publications

References 52 publications

PRKDC mutations in a SCID patient with profound neurological abnormalities

PRKDC mutations in a SCID patient with profound neurological abnormalities

A DNA-PKcs mutation in a radiosensitive T–B– SCID patient inhibits Artemis activation and nonhomologous end-joining

DNA strand break repair and neurodegeneration

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