N.BstNBI is a nicking endonuclease that recognizes the sequence GAGTC and nicks the top strand preferentially. The Type IIs restriction endonucleases PleI and MlyI also recognize GAGTC, but cleave both DNA strands. Cloning and sequencing the genes encoding each of these three endonucleases discloses significant sequence similarities. Mutagenesis studies reveal a conserved set of catalytic residues among the three endonucleases, suggesting that they are closely related to each other. Furthermore, PleI and MlyI contain a single active site for DNA cleavage. The results from cleavage assays show that the reactions catalyzed by PleI and MlyI are sequential two step processes. The double-stranded DNA is first nicked on one DNA strand and then further cleaved on the second strand to form linear DNA. Gel filtration analysis shows that MlyI dimerizes in the presence of a cognate DNA and Ca(2+) whereas N.BstNBI remains a monomer, implicating dimerization as a requisite for the second strand cleavage. We suggest that N.BstNBI, MlyI and PleI diverged from a common ancestor and propose that N.BstNBI differs from MlyI and PleI in having an extremely limited second strand cleavage activity, resulting in a site-specific nicking endonuclease.

The serotype F phage w42 of Staphylococcus aureus is a triple-converting bacteriophage that encodes the staphylokinase gene (sak) and the enterotoxin A gene (entA). Lysogeny results in loss of expression of the chromosomal b-haemolysin gene (hlb) (negative conversion), the expression of staphylokinase and enterotoxin A (positive conversion), and the acquisition of resistance to lysis by all 23 phages of the International Basic Set (IBS) of S. aureus typing phages. Until this study, the basis of w42 resistance to lysis by exogenous phages was unknown. The authors report here that phage w42 encodes a restriction-modification (R-M) system, termed Sau42I, adjacent to and in the same orientation to the phage integrase gene int. The genes encoding Sau42I were cloned and sequenced, and found to consist of two overlapping reading frames, ORF S (specificity) and ORF RM (restriction-modification), in the same orientation. The ORFs share a high degree of DNA and amino acid sequence homology with the previously characterized BcgI R-M system of Bacillus coagulans. Expression of the cloned Sau42I ORF S and ORF RM in S. aureus 80CR3 transformants from a plasmid vector conferred resistance to lysis by all 23 IBS phages. Similarly, transformants of S. aureus RN4220 harbouring recombinant plasmids containing both ORFs were resistant to lysis by the IBS typing phages. However, transformants harbouring plasmids encoding either ORF S or ORF RM were susceptible to lysis by the IBS phages, and they had the same phage-susceptibility pattern as the respective parental isolates. In vitro analysis of crude and partially purified extracts of S. aureus transformants harbouring both the w42 ORF S and ORF RM genes indicated that Sau42I has endonuclease activity and requires co-factors Mg 2+ and S-adenosylmethionine in order to function, and activity is optimized at pH 8, although the precise recognition sequence has yet to be determined. The findings of this study confirm that w42 is a quadruple-converting phage, believed to be the first described for S. aureus, and show that it encodes a novel R-M system termed Sau42I.

Background Helicobacter pylori is the etiologic agent of common gastritis and a risk factor for gastric cancer. It is also one of the richest sources of Type II restriction-modification (R-M) systems in microorganisms.Principal FindingsWe have cloned, expressed and purified a new restriction endonuclease HpyAV from H. pylori strain 26695. We determined the HpyAV DNA recognition sequence and cleavage site as CCTTC 6/5. In addition, we found that HpyAV has a unique metal ion requirement: its cleavage activity is higher with transition metal ions than in Mg++. The special metal ion requirement of HpyAV can be attributed to the presence of a HNH catalytic site similar to ColE9 nuclease instead of the canonical PD-X-D/EXK catalytic site found in many other REases. Site-directed mutagenesis was carried out to verify the catalytic residues of HpyAV. Mutation of the conserved metal-binding Asn311 and His320 to alanine eliminated cleavage activity. HpyAV variant H295A displayed approximately 1% of wt activity.Conclusions/SignificanceSome HNH-type endonucleases have unique metal ion cofactor requirement for optimal activities. Homology modeling and site-directed mutagenesis confirmed that HpyAV is a member of the HNH nuclease family. The identification of catalytic residues in HpyAV paved the way for further engineering of the metal binding site. A survey of sequenced microbial genomes uncovered 10 putative R-M systems that show high sequence similarity to the HpyAV system, suggesting lateral transfer of a prototypic HpyAV-like R-M system among these microorganisms.

Background: Screening newborns for severe combined immunodeficiency (SCID) aims for early identification and treatment of the affected newborns. Adenosine deaminase (ADA) deficiency, a defect in the purine metabolic pathway, is a major cause of SCID and is characterized by the accumulation of adenosine (Ado) and deoxyadenosine (dAdo) in dried blood spots (DBSs). If left untreated, infants with this disorder are at risk of life-threatening infections. Analysis of T-cell receptor excision circles (TRECs) in DBS samples is the gold-standard screening method. However, TREC analysis is insufficient to determine SCID etiology, and a fraction of ADA-SCID may not be detected.