Arunas Lagunavicius

Novel Subtype of Type IIs Restriction Enzymes BfiI ENDONUCLEASE EXHIBITS SIMILARITIES TO THE EDTA-RESISTANT NUCLEASE Nuc OF SALMONELLA TYPHIMURIUM

The type IIs restriction enzyme BfiI recognizes the non-palindromic nucleotide sequence 5′-ACTGGG-3′ and cleaves complementary DNA strands 5/4 nucleotides downstream of the recognition sequence. The genes coding for the BfiI restriction-modification (R-M) system were cloned/sequenced and biochemical characterization of BfiI restriction enzyme was performed. The BfiI R-M system contained three proteins: two N4-methylcytosine methyltransferases and a restriction enzyme. Sequencing of bisulfite-treated methylated DNA indicated that each methyltransferase modifies cytosines on opposite strands of the recognition sequence. The N-terminal part of the BfiI restriction enzyme amino acid sequence revealed intriguing similarities to an EDTA-resistant nuclease of Salmonella typhimurium. Biochemical analyses demonstrated that BfiI, like the nuclease of S. typhimurium, cleaves DNA in the absence of Mg2+ ions and hydrolyzes an artificial substrate bis(p-nitrophenyl) phosphate. However, unlike the nonspecific S. typhimurium nuclease, BfiI restriction enzyme cleaves DNA specifically. We propose that the DNA-binding specificity of BfiI stems from the C-terminal part of the protein. The catalytic N-terminal subdomain ofBfiI radically differs from that of type II restriction enzymes and is presumably similar to the EDTA-resistant nonspecific nuclease of S. typhimurium; therefore, BfiI did not require metal ions for catalysis. We suggest that BfiI represents a novel subclass of type IIs restriction enzymes that differs from the archetypal FokI endonuclease by the fold of its cleavage domain, the domain location, and reaction mechanism.

The Metal-independent Type IIs Restriction Enzyme BfiI is a Dimer that Binds Two DNA Sites but has Only One Catalytic Centre

BfiI is a novel type IIs restriction endonuclease that, unlike all other restriction enzymes characterised to date, cleaves DNA in the absence of Mg(2+). The amino acid sequence of the N-terminal part of BfiI has some similarities to Nuc of Salmonella typhimurium, an EDTA-resistant nuclease akin to phospholipase D. The dimeric form of Nuc contains a single active site composed of residues from both subunits. To examine the roles of the amino acid residues of BfiI that align with the catalytic residues in Nuc, a set of alanine replacement mutants was generated by site-directed mutagenesis. The mutationally altered forms of BfiI were all catalytically inactive but were still able to bind DNA specifically. The active site of BfiI is thus likely to be similar to that of Nuc. BfiI was also found by gel-filtration to be a dimer in solution. Both gel-shift and pull-down assays indicated that the dimeric form of BfiI binds two copies of its recognition sequence. In reactions on plasmids with either one or two copies of its recognition sequence, BfiI cleaved the DNA with two sites more rapidly than that with one site. Yet, when bound to two copies of its recognition sequence, the BfiI dimer cleaved only one phosphodiester bond at a time. The dimer thus seems to contain two DNA-binding domains but only one active site.

Photocaged variants of the MunI and PvuII restriction enzymes.

Regulation of proteins by light is a new and promising strategy for the external control of biological processes. In this study, we demonstrate the ability to regulate the catalytic activity of the MunI and PvuII restriction endonucleases with light. We used two different approaches to attach a photoremovable caging compound, 2-nitrobenzyl bromide (NBB), to functionally important regions of the two enzymes. First, we covalently attached a caging molecule at the dimer interface of MunI to generate an inactive monomer. Second, we attached NBB at the DNA binding site of the single-chain variant of PvuII (scPvuII) to prevent binding and cleavage of the DNA substrate. Upon removal of the caging group by UV irradiation, nearly 50% of the catalytic activity of MunI and 80% of the catalytic activity of PvuII could be restored.

Efficient gene transfection using novel cationic polymers poly(hydroxyalkylene imines).

A series of novel cationic polymers poly(hydroxyalkylene imines) were synthesized and tested for their ability to transfect cells in vitro and in vivo. Poly(hydroxyalkylene imines), in particular, poly(2-hydroxypropylene imine) (pHP), poly(2-hydroxypropylene imine ethylene imine) (pHPE), and poly(hydroxypropylene imine propylene imine) (pHPP) were synthesized by polycondensation reaction from 1,3-diamino-2-propanol and the appropriate dibromide. Electron microscopic examination demonstrated that the resulting polymers condensed DNA into toroid shape complexes of 100-150 nm in size. Transfection studies showed that all three polymers were able to deliver genetic material into the cell, with pHP being superior to pHPP and pHPE. pHP acted as an efficient gene delivery agent in a variety of different cell lines and outcompeted most of the widely used polymer or lipid based transfection reagents. Intravenous administration of pHP-DNA polyplexes in mice followed by the reporter gene analysis showed that the reagent was suitable for in vivo applications. In summary, the results indicate that pHP is a new efficient reagent for gene delivery in vitro and in vivo.