SgrAI is a type II restriction endonuclease that cuts an unusually long recognition sequence and exhibits allosteric self-modulation of DNA activity and sequence specificity. Precleaved primary site DNA has been shown to be an allosteric effector [Hingorani-Varma & Bitinaite, (2003) J. Biol. Chem. 278, 40392-40399], stimulating cleavage of both primary (CR|CCGGYG, | indicates cut site, R=A,G, Y=C,T) and secondary (CR|CCGGY(A/C/T) and CR|CCGGGG) site DNA sequences. The fact that DNA is the allosteric effector of this endonuclease suggests at least two DNA binding sites on the functional SgrAI molecule, yet crystal structures of SgrAI [Dunten, et al., (2008) Nucleic Acids Res. 36, 5405–5416] show only one DNA duplex bound to one dimer of SgrAI. We show that SgrAI forms species larger than dimers or tetramers (High Molecular Weight Species, HMWS) in the presence of sufficient concentrations of SgrAI and its primary site DNA sequence, that are dependent on the concentration of the DNA bound SgrAI dimer. Analytical ultracentrifugation indicates that the HMWS is heterogeneous, has sedimentation coefficients of 15–20 s, and is composed of possibly 4–12 DNA bound SgrAI dimers. SgrAI bound to secondary site DNA will not form HMWS itself, but can bind to HMWS formed with primary site DNA and SgrAI. Uncleaved, as well as precleaved, primary site DNA is capable of stimulating HMWS formation. Stimulation of DNA cleavage by SgrAI, at primary as well as secondary sites, is also dependent on the concentration of primary site DNA (cleaved or uncleaved) bound SgrAI dimers. SgrAI bound to secondary site DNA does not have significant stimulatory activity. We propose that the oligomers of DNA bound SgrAI (i.e. HMWS) are the activated, or activatable, form of the enzyme.

BackgroundSequential compression devices (SCDs) are commonly used for thromboprophylaxis in postoperative patients but compliance is often poor. We investigated causes for noncompliance, examining both hospital and patient related factors.Methods100 patients undergoing inpatient urologic surgery were enrolled. All patient had SCD sleeves placed preoperatively. Postoperative observations determined SCD compliance and reasons for non-compliance. Patient demographics, length of stay, inpatient unit type, and surgery type were recorded. At discharge, a patient survey gauged knowledge and attitudes regarding SCDs and bother with SCDs. Statistical analysis was performed to correlate SCD compliance with patient demographics; patient knowledge and attitudes regarding SCDs; and patient self-reported bother with SCDs.ResultsObserved overall compliance was 78.6%. The most commonly observed reasons for non-compliance were SCD machines not being initially available on the ward (71% of non-compliant observations on post-operative day 1) and SCD use not being restarted promptly after return to bed (50% of non-compliant observations for entire hospital stay). Mean self-reported bother scores related to SCDs were low, ranging from 1–3 out of 10 for all 12 categories of bother assessed. Patient demographics, knowledge, attitudes and bother with SCD devices were not significantly associated with non-compliance.ConclusionsPatient self-reported bother with SCD devices was low. Hospital factors, including SCD machine availability and timely restarting of devices by nursing staff when a patient returns to bed, played a greater role in SCD non-compliance than patient factors. Identifying and addressing hospital related causes for poor SCD compliance may improve postoperative urologic patient safety.

DNA repair is fundamental to genome stability and is found in all three domains of life. However, many archaeal species, such as Methanopyrus kandleri, contain only a subset of the eukaryotic nucleotide excision repair (NER) homologues, and those present often contain significant differences compared to their eukaryotic homologues. To clarify the role of the NER XPG-like protein Mk0566 from M. kandleri, its biochemical activity and three dimensional structure were investigated. Both were found to be more similar to human FEN-1 than human XPG, suggesting a biological role in replication and long-patch base excision repair rather than in NER.