The restriction endonuclease (REase) R.KpnI is an orthodox Type IIP enzyme, which binds to DNA in the absence of metal ions and cleaves the DNA sequence 5'-GGTAC--C-3' in the presence of Mg2+ as shown generating 3' four base overhangs. Bioinformatics analysis reveals that R.KpnI contains a betabetaalpha-Me-finger fold, which is characteristic of many HNH-superfamily endonucleases, including homing endonuclease I-HmuI, structure-specific T4 endonuclease VII, colicin E9, sequence non-specific Serratia nuclease and sequence-specific homing endonuclease I-PpoI. According to our homology model of R.KpnI, D148, H149 and Q175 correspond to the critical D, H and N or H residues of the HNH nucleases. Substitutions of these three conserved residues lead to the loss of the DNA cleavage activity by R.KpnI, confirming their importance. The mutant Q175E fails to bind DNA at the standard conditions, although the DNA binding and cleavage can be rescued at pH 6.0, indicating a role for Q175 in DNA binding and cleavage. Our study provides the first experimental evidence for a Type IIP REase that does not belong to the PD...D/EXK superfamily of nucleases, instead is a member of the HNH superfamily.

Actin-related protein Arp8 is a component of the INO80 chromatin remodeling complex. Yeast Arp8 (yArp8) comprises two domains: a 25-KDa N-terminal domain, found only in yeast, and a 75-KDa C-terminal domain (yArp8CTD) that contains the actin fold and is conserved across other species. The crystal structure shows that yArp8CTD contains three insertions within the actin core. Using a combination of biochemistry and EM, we show that Arp8 forms a complex with nucleosomes, and that the principal interactions are via the H3 and H4 histones, mediated through one of the yArp8 insertions. We show that recombinant yArp8 exists in monomeric and dimeric states, but the dimer is the biologically relevant form required for stable interactions with histones that exploits the twofold symmetry of the nucleosome core. Taken together, these data provide unique insight into the stoichiometry, architecture, and molecular interactions between components of the INO80 remodeling complex and nucleosomes, providing a first step toward building up the structure of the complex.histone exchange | ATPase C hromatin remodeling complexes have recognized roles in remodeling of nucleosomes during transcription and DNA repair (1). In addition to the DNA translocase subunit, these complexes typically consist of several proteins, although the biochemical functions of these additional subunits remain a mystery in most cases. Actin-related proteins (ARPs) are a group of proteins with homology to actin (2). Several ARP family members (Arp4-Arp9) are components of nucleosome-modifying complexes, including remodelers (e.g., INO80, RSC), histone exchangers (e.g., INO80, Swr1), and acetylation complexes (e.g., NuA4, Tip60) (1, 3). ARPs with known structures are Arp2 and Arp3 within the cytoplasmic Arp2/3 complex (4) and nuclear Arp4 (5). Nuclear ARPs have diverged further from the actin progenitor than Arp2/ 3, and all contain large insertions in the actin-like fold that have unknown functions (2). A regulatory role has been suggested for nuclear ARPs (6, 7), but this remains poorly understood. More recently, roles for nuclear ARPs besides chromatin remodeling have been suggested (8).Arp8 contains a conserved domain of ∼630 amino acids that includes a core of ∼390 amino acids with homology to actin and three large insertions of unknown function (SI Appendix, Fig. S1). Arp8 is essential for activity of INO80, and deletion of Arp8 results in loss of INO80 function, with multiple effects on such processes as double-strand break repair (9), homologous recombination (10), and chromosome alignment (11). Yeast strains lacking Arp8 fail to recruit Arp4 and actin to the INO80 complex, suggesting associations among these proteins within the complex (12).EM studies have been carried out on SWI/SNF, RSC, and ACF complexes (13)(14)(15)(16)(17). Although these studies have revealed the overall architecture of the complexes, the mechanism of remodeling remains unclear. Crystal structures have been determined in very few of the individual protein subunits of chromatin re...

Quantitative evaluation of small molecule permeation and accumulation in Gram-negative bacteria is important for drug development against these bacteria. While these measurements are commonly performed at physiological pH, Escherichia coli and many other Enterobacteriaceae infect human gastrointestinal and urinary tracts, where they encounter different pH conditions. To understand how external pH affects permeation and accumulation of small molecules in E. coli cells, we apply second harmonic generation (SHG) spectroscopy using SHG-active antimicrobial compound malachite green as the probe molecule. Using SHG, we quantify periplasmic and cytoplasmic accumulations separately in live E. coli cells, which was never done before. Compartment-wise measurements reveal accumulation of the probe molecule in cytoplasm at physiological and alkaline pH, while entrapment in periplasm at weakly acidic pH and retention in external solution at highly acidic pH. Behind such disparity in localizations, up to 2 orders of magnitude reduction in permeability across the inner membrane at weakly acidic pH and outer membrane at highly acidic pH are found to play key roles. Our results unequivocally demonstrate the control of external pH over entry and compartment-wise distribution of small molecules in E. coli cells, which is a vital information and should be taken into account in antibiotic screening against E. coli and other Enterobacteriaceae members. In addition, our results demonstrate the ability of malachite green as an excellent SHG-indicator of changes of individual cell membrane and periplasm properties of live E. coli cells in response to external pH change from acidic to alkaline. This finding, too, has great importance, as there is barely any other molecular probe that can provide similar information.

The characteristic feature of type II restriction endonucleases (REases) is their exquisite sequence specificity and obligate Mg 2؉ requirement for catalysis. Efficient cleavage of DNA only in the presence of Ca 2؉ ions, comparable with that of Mg 2؉ , is previously not described. Most intriguingly, KpnI REase exhibits Ca 2؉ -dependent specific DNA cleavage. Moreover, the enzyme is highly promiscuous in its cleavage pattern on plasmid DNAs in the presence of Mn 2؉ or Mg 2؉ , with the complete suppression of promiscuous activity in the presence of Ca 2؉ . KpnI methyltransferase does not exhibit promiscuous activity unlike its cognate REase. The REase binds to oligonucleotides containing canonical and mapped noncanonical sites with comparable affinities. However, the extent of cleavage is varied depending on the metal ion and the sequence. The ability of the enzyme to be promiscuous or specific may reflect an evolutionary design. Based on the results, we suggest that the enzyme KpnI represents an REase evolving to attain higher sequence specificity from an ancient nonspecific nuclease.Type II REases recognize specific DNA sequences, which vary from 4 to 8 bp and cleave both the strands in Mg 2ϩ -dependent reaction. They exhibit remarkable features in discriminating between specific and nonspecific DNA, not only in DNA binding but also during DNA cleavage (1). Based on the divalent metal ion requirement for DNA binding, REases have been classified into two groups (2). For example, EcoRI, BamHI, RsrI, and a few other enzymes bind to DNA preferentially at their recognition sites in the absence of divalent metal ions (3-5). In contrast, EcoRV, PvuII, TaqI, and a few other enzymes bind to cognate sequences in a divalent metal ion-dependent manner (6 -8). However, both the groups of enzymes need Mg 2ϩ for catalysis. KpnI REase isolated from Klebsiella pneumoniae recognizes the palindromic double-stranded DNA sequence 5Ј-GGTAC2C-3Ј and cleaves DNA by generating a 3Ј, 4-base overhang (18). The enzyme binds to specific DNA in the absence of divalent metal ions (19). In this paper, we describe the highly promiscuous cleavage of DNA in the presence of Mg 2ϩ and Mn 2ϩ . The promiscuous activity is completely suppressed in the presence of Ca 2ϩ , which induces site-specific DNA cleavage. EXPERIMENTAL PROCEDURESEnzymes and DNA-KpnI REase was purified as described previously (18). The enzyme was diluted in binding buffer (20 mM Tris-HCl (pH 7.4), 25 mM NaCl, and 5 mM 2-mercaptoethanol) for all the studies. The concentration of the enzyme was estimated by the method of Bradford (20). One unit of KpnI REase is defined as the amount of enzyme required for complete digestion of 1 g of DNA at 37°C for 1 h by using assay buffer containing 5 mM Mg 2ϩ . T4 polynucleotide kinase and Klenow polymerase were purchased from New England Biolabs. Oligonucleotides were from Microsynth Inc., Switzerland, and purified on 18% urea-polyacrylamide gel (21). The purified oligonucleotides were end-labeled with T4 polynucleotide kinase and [␥-32 P]ATP ...

KpnI REase recognizes palindromic sequence, GGTAC↓C, and forms complex in the absence of divalent metal ions, but requires the ions for DNA cleavage. Unlike most other REases, R.KpnI shows promiscuous DNA cleavage in the presence of Mg2+. Surprisingly, Ca2+ suppresses the Mg2+-mediated promiscuous activity and induces high fidelity cleavage. To further analyze these unique features of the enzyme, we have carried out DNA binding and kinetic analysis. The metal ions which exhibit disparate pattern of DNA cleavage have no role in DNA recognition. The enzyme binds to both canonical and non-canonical DNA with comparable affinity irrespective of the metal ions used. Further, Ca2+-imparted exquisite specificity of the enzyme is at the level of DNA cleavage and not at the binding step. With the canonical oligonucleotides, the cleavage rate of the enzyme was comparable for both Mg2+- and Mn2+-mediated reactions and was about three times slower with Ca2+. The enzyme discriminates non-canonical sequences poorly from the canonical sequence in Mg2+-mediated reactions unlike any other Type II REases, accounting for the promiscuous behavior. R.KpnI, thus displays properties akin to that of typical Type II REases and also endonucleases with degenerate specificity in its DNA recognition and cleavage properties.