Phosphorylation of DNA-PKcs at the S2056 cluster ensures efficient and productive lymphocyte development in XLF-deficient mice.

The nonhomologous end-joining (NHEJ) pathway is a major DNA double-strand break repair pathway in mammals and is essential for lymphocyte development. Ku70 and Ku80 heterodimer (KU) initiates NHEJ, thereby recruiting and activating the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). While DNA-PKcs deletion only moderately impairs end-ligation, the expression of kinase-dead DNA-PKcs completely abrogates NHEJ. Active DNA-PK phosphorylates DNA-PKcs at two clusters-PQR around S2056 (S2053 in mouse) and ABCDE around T2609. Alanine substitution at the S2056 cluster moderately compromises end-ligation on plasmid-based assays. But, mice carrying alanine substitution at all five serine residues within the S2056 cluster (DNA-PKcsPQR/PQR) display no defect in lymphocyte development, leaving the physiological significance of S2056 cluster phosphorylation elusive. Xlf is a nonessential NHEJ factor. Xlf -/- mice have substantial peripheral lymphocytes that are completely abolished by the loss of DNA-PKcs, the related ATM kinases, other chromatin-associated DNA damage response factors (e.g., 53BP1, MDC1, H2AX, and MRI), or RAG2-C-terminal regions, suggesting functional redundancy. While ATM inhibition does not further compromise end-ligation, here we show that in XLF-deficient background, DNA-PKcs S2056 cluster phosphorylation is critical for normal lymphocyte development. Chromosomal V(D)J recombination from DNA-PKcsPQR/PQRXlf -/- B cells is efficient but often has large deletions that jeopardize lymphocyte development. Class-switch recombination junctions from DNA-PKcsPQR/PQRXlf -/- mice are less efficient and the residual junctions display decreased fidelity and increased deletion. These findings establish a role for DNA-PKcs S2056 cluster phosphorylation in physiological chromosomal NHEJ, implying that S2056 cluster phosphorylation contributes to the synergy between XLF and DNA-PKcs in end-ligation.

Norwalk-like virus 95/96-US strain is a major cause of gastroenteritis outbreaks in Australia.

Norwalk-like viruses (NLVs) were detected using a nested reverse transcriptase-polymerase chain reaction (RT-PCR) with primers directed to the RNA polymerase region. Samples were examined from 11 separate outbreaks of gastroenteritis and five sporadic cases of childhood gastroenteritis between 1997 and 2000. Phylogenetic analysis of the 298 bp sequences showed that all strains belong to NLV genogroup II and the majority of the sequenced isolates (30/36) were members of the 95/96-US subset of strains associated with outbreaks recorded worldwide between 1995 and 1996. This was confirmed by analysis of the full length capsid region of a representative Australian isolate. This study demonstrates the usefulness of targeting primers for NLVs to the predominant circulating genotype(s) and confirms the spread of this subtype globally, including the Southern Hemisphere.