DNA and spermidine provide a switch mechanism to regulate the activity of restriction enzyme Nae I.

Abstract

Sequence-specific DNA-protein interactions are basic to DNA function. To better understand these interactions, we studied the effect of position on cleavage of DNA by the type II restriction enzyme (EC 3.1.21.4) Nae I. We discovered two classes of Nae I restriction sites: sites susceptible and sites resistant to cleavage. Kinetic analysis showed that Nae I was activated by DNA containing cleavable Nae I sites to rapidly cleave resistant Nae I sites by a noncompetitive mechanism with a Km for substrate DNA of about 2 nM and a KA for activating DNA of about 6 nM; activation increased catalysis but not substrate binding. Deletion mutagenesis in vitro showed that sequences flanking the Nae I recognition site were responsible for the differences between activating and nonactivating Nae I sites. The polyamine spermidine had a dramatic effect on the interaction of Nae I with DNA; in the presence of 1 mM spermidine, resistant sites were cleaved rapidly and cleavable DNA inhibited cleavage. The direct regulation of enzymatic activity by DNA sequences in trans, and the modulation of this regulation by a polyamine that is sensitive to the cell cycle, provides a regulatory switch mechanism. The implications of this switch for biological control functions are discussed.The type II restriction/modification systems (1), like the repressors (2), are important model systems for sequencespecific DNA-protein interactions that are basic to many biological processes. In the course of studying these interactions, we made the quite unexpected discovery that cleavage by the type II restriction enzyme Nae I can be controlled by specific DNA sequences; the ability to cleave and the action of external DNA sequences were in turn dramatically affected by spermidine. Nae I is isolated from Nocardia aerocolonigenes, a member of a common family, Nocardiaceae, of soil actinomycetes. It cleaves within the sequence GCC/GGC (ref. 3 and supplier catalogs) and, like other type II enzymes, requires only Mg2+ for activity and exhibits only a single function, cleavage. Using commercially available Nae I, we found DNA sequences containing Nae I recognition sites that were cleaved rapidly and Nae I sites that were almost totally resistant to cleavage. Studies of Nae I interaction with these sites showed that Nae I was activated to rapidly cleave resistant sites and that the activator was a cleavable Nae I site. The activated cleavage reaction followed MichaelisMenten kinetics, which indicated that activation was noncompetitive and worked by increasing catalysis rather than by increasing the affinity of the enzyme for its substrate. Deletion mutagenesis in vitro showed that sequences flanking the Nae I recognition site were responsible for the enzymeregulatory properties of the cleavable sites. Spermidine could reverse the effect; in the presence of 1 mM spermidine, resistant sites were cleaved rapidly but cleavable DNA inhibited their cleavage, providing a regulatory switch mechanism. MATERIALS AND METHODSRestriction enzyme cleavage reactions w...

Kinetic Analysis of the Coordinated Interaction of SgrAI Restriction Endonuclease with Different DNA Targets

“…For example, Type IIE endonucleases such as NaeI and EcoRII are homodimeric proteins that have two distinct DNA binding clefts (18 -20) and thus are capable of binding two copies of their recognition sequence per protein dimer. However, only one of these recognition sequences is cleaved while the other serves as an allosteric effector (21)(22)(23)(24)(25)(26)(27)(28). Studies with NaeI reveal that the recognition sequences flanked by G/C-rich regions are preferred by the activator site, whereas the catalytic site prefers A/T-rich regions (22,25).…”

“…However, only one of these recognition sequences is cleaved while the other serves as an allosteric effector (21)(22)(23)(24)(25)(26)(27)(28). Studies with NaeI reveal that the recognition sequences flanked by G/C-rich regions are preferred by the activator site, whereas the catalytic site prefers A/T-rich regions (22,25). In the case of EcoRII, it has been shown that some non-cleavable or pre-digested recognition sequences can be bound at the activator site and stimulate the cleavage of refractory sites (27).…”

Sau3AI, a Monomeric Type II Restriction Endonuclease That Dimerizes on the DNA and Thereby Induces DNA Loops

“…FokI (3,4); the dimeric type IIe enzymes, e.g. EcoRII and NaeI, which require binding to a second recognition site for cleavage (5,6); dimeric restriction endonucleases like SgrAI, which also require two recognition sites for activity and tetramerize on the DNA (7); and tetrameric type IIf restriction endonucleases like SfiI, Cfr10I, and NgoMIV (8 -10), which interact with two recognition sites, loop out the DNA, and cleave the two sites in a concerted manner. Structural information is available for FokI (4,11), NaeI (12), Cfr10I (9), and NgoMIV (10), in addition to structural information on the orthodox homodimeric type II restriction endonucleases BamHI, BglI, BglII, BsoBI, EcoRI, EcoRV, MunI, and PvuII (for review, see Ref.…”

Selective inhibition of cytosine‐DNA methylases by polyamines

“…DNA concentrations are expressed as/.tg per/ll. stimulatory effect of spermidine on the restriction enzyme NaeI, mainly in the absence of activating oligonucleotides [26].…”