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SummaryCtIP is involved in the resection of double-stranded DNA breaks during the S and G2 phases of the cell cycle for repair by homologous recombination. Acting in concert with the MRN complex, it plays a particularly important role in handling complex DNA end structures by localised nucleolytic processing of DNA termini in preparation for longer range resection. Here we show that human CtIP is a tetrameric protein adopting a dumbbell architecture in which DNA binding domains are connected by long coiled-coils. The protein complex binds two short DNA duplexes with high affinity and bridges DNA molecules in trans. DNA binding is potentiated by dephosphorylation and is not specific for DNA end structures per se. However, the affinity for linear DNA molecules is increased if the DNA terminates with more complex structures including forked ssDNA overhangs and model nucleoprotein conjugates. This work provides a biochemical and structural basis for the function of CtIP at complex DNA breaks.

Streptococcus pyogenes nuclease A (SpnA) is a recently discovered DNase that plays a role in virulence as shown in a mouse infection model. SpnA is the only cell wall-anchored DNase found in S. pyogenes thus far and shows a unique protein architecture. The C-terminal nuclease domain contains highly conserved catalytic site and Mg(2+) binding site residues. However, expression of the SpnA nuclease domain alone resulted in a soluble, but enzymatically inactive protein. We found that at least two out of three oligonucleotide/oligosaccharide-binding fold motifs found in the N-terminal domain are required for SpnA activity, probably contributing to substrate binding. Using a combination of a spnA deletion mutant and a Lactococcus lactis'gain-of-function' mutant, we have shown that SpnA promotes survival in whole human blood and in neutrophil killing assays and this is, at least in part, achieved by the destruction of neutrophil extracellular traps (NETs). We observed higher frequencies for anti-SpnA antibodies in streptococcal disease patient sera (79%, n = 19) compared with sera from healthy donors (33%, n = 9) suggesting that SpnA is expressed during infection. Detection of anti-SpnA antibodies in patient serum might be useful for the diagnostic of post-streptococcal diseases, such as acute rheumatic fever or glomerulonephritis.

Streptococcus pyogenes nuclease A (SpnA) is a recently discovered DNase that plays a role in virulence as shown in a mouse infection model. SpnA is the only cell wall-anchored DNase found in S. pyogenes thus far and shows a unique protein architecture. The C-terminal nuclease domain contains highly conserved catalytic site and Mg(2+) binding site residues. However, expression of the SpnA nuclease domain alone resulted in a soluble, but enzymatically inactive protein. We found that at least two out of three oligonucleotide/oligosaccharide-binding fold motifs found in the N-terminal domain are required for SpnA activity, probably contributing to substrate binding. Using a combination of a spnA deletion mutant and a Lactococcus lactis'gain-of-function' mutant, we have shown that SpnA promotes survival in whole human blood and in neutrophil killing assays and this is, at least in part, achieved by the destruction of neutrophil extracellular traps (NETs). We observed higher frequencies for anti-SpnA antibodies in streptococcal disease patient sera (79%, n = 19) compared with sera from healthy donors (33%, n = 9) suggesting that SpnA is expressed during infection. Detection of anti-SpnA antibodies in patient serum might be useful for the diagnostic of post-streptococcal diseases, such as acute rheumatic fever or glomerulonephritis.

Proteins displayed on the cell surfaces of pathogenic organisms are the front-line troops of bacterial attack, playing critical roles in colonization, infection and virulence. Although such proteins can often be recognized from genome sequence data, through characteristic sequence motifs, their functions are often unknown. One such group of surface proteins is attached to the cell surface of Gram-positive pathogens through the action of sortase enzymes. Some of these proteins are now known to form pili: long filamentous structures that mediate attachment to human cells. Crystallographic analyses of these and other cell surface proteins have uncovered novel features in their structure, assembly and stability, including the presence of inter- and intramolecular isopeptide crosslinks. This improved understanding of structures on the bacterial cell surface offers opportunities for the development of some new drug targets and for novel approaches to vaccine design.