Phage T4 endonuclease II (EndoII), a GIY-YIG endonuclease lacking a carboxy-terminal DNA-binding domain, was subjected to site-directed mutagenesis to investigate roles of individual amino acids in substrate recognition, binding, and catalysis. The structure of EndoII was modeled on that of UvrC. We found catalytic roles for residues in the putative catalytic surface (G49, R57, E118, and N130) similar to those described for I-TevI and UvrC; in addition, these residues were found to be important for substrate recognition and binding. The conserved glycine (G49) and arginine (R57) were essential for normal sequence recognition. Our results are in agreement with a role for these residues in forming the DNA-binding surface and exposing the substrate scissile bond at the active site. The conserved asparagine (N130) and an adjacent proline (P127) likely contribute to positioning the catalytic domain correctly. Enzymes in the EndoII subfamily of GIY-YIG endonucleases share a strongly conserved middle region (MR, residues 72 to 93, likely helical and possibly substituting for heterologous helices in I-TevI and UvrC) and a less strongly conserved N-terminal region (residues 12 to 24). Most of the conserved residues in these two regions appeared to contribute to binding strength without affecting the mode of substrate binding at the catalytic surface. EndoII K76, part of a conserved NUMOD3 DNA-binding motif of homing endonucleases found to overlap the MR, affected both sequence recognition and catalysis, suggesting a more direct involvement in positioning the substrate. Our data thus suggest roles for the MR and residues conserved in GIY-YIG enzymes in recognizing and binding the substrate.Endonuclease II (EndoII) of coliphage T4, encoded by gene denA, catalyzes the initial step in host DNA degradation. It causes irreversible host shutoff and also initiates a nucleotide scavenge pathway that provides precursors for phage DNA synthesis (8). T4 phage DNA is protected from EndoII by the substitution of 5-hydroxymethyl deoxycytosine for dC during T4 DNA synthesis (9, 43). EndoII shares the sequence elements defining the GIY-YIG family of proteins (13,20) that includes the repair endonuclease UvrC and many homing endonucleases, such as the intron-encoded T4 endonuclease ITevI (20) and I-BmoI (10), as well as some type II restriction endonucleases (2,19). Like the homing endonucleases in the GIY-YIG family, EndoII cleaves a long, asymmetric, and ambiguous DNA sequence (4-6, 21). However, in contrast to what has been found for I-TevI (24), EndoII cleaves the two strands independently of each other (7), and only a CG dinucleotide 2 bp away from the scissile bond is strongly conserved (5). Double-strand cleavage by EndoII is the result of concerted singlestrand nicks (7), but many positions are only nicked, not cleaved.In UvrC (44), as well as in the GIY-YIG homing endonucleases (10, 12), the conserved motifs and catalytic activity reside in the amino-terminal domain, but the primary binding energy is conferred by a carboxy-termin...

SummaryThe 136 codon (408 bp) denA gene encoding endonuclease II (EndoII) of bacteriophage T4 was unambiguously identi®ed through sequencing and subsequent cloning. EndoII prepared from cloned DNA through coupled in vitro transcription±translation nicked and cut DNA in vitro in a sequence-speci®c manner. In vitro (and in vivo ), the bottom strand was nicked between the ®rst and second base pair to the right of a topstrand CCGC motif shared by favoured in vitro and in vivo cleavage sites; top-strand cleavage positions varied. To the right of the cleavage position, favoured in vitro sites lacked a sequence element conserved at favoured in vivo sites. In pBR322 DNA, the sites cleaved in vivo as previously described were also cleaved in vitro, but in vitro additional sites were nicked or cleaved and the preference for individual sites was different. Also, different from the in vivo reaction, nicking was more frequent than ds cutting; in many copies of a ds cleavage site, only the bottom strand was nicked in vitro. A model is discussed in which sequential nicking of the two strands, and different factors in¯u-encing bottom-strand nicking and top-strand nicking, can explain the differences between the in vitro and the in vivo reaction.

Synthetic sites inserted into a plasmid were used to analyze the sequence requirements for in vivo DNA cleavage dependent on bacteriophage T4 endonuclease II. A 16-bp variable sequence surrounding the cleavage site was sufficient for cleavage, although context both within and around this sequence influenced cleavage efficiency. The most efficiently cleaved sites matched the sequence CGRCCGCNTTGGCNGC, in which the strongly conserved bases to the left were essential for cleavage. The less-conserved bases in the center and in the right half determined cleavage efficiency in a manner not directly correlated with the apparent base preference at each position; a sequence carrying, in each of the 16 positions, the base most preferred in natural sites in pBR322 was cleaved infrequently. This, along with the effects of substitutions at one or two of the lessconserved positions, suggests that several combinations of bases can fulfill the requirements for recognition of the right part of this sequence. The replacements that improve cleavage frequency are predicted to influence helical twist and roll, suggesting that recognition of sequence-dependent DNA structure and recognition of specific bases are both important. Upon introduction of a synthetic site, cleavage at natural sites within 800 to 1,500 bp from the synthetic site was significantly reduced. This suggests that the enzyme may engage more DNA than its cleavage site and cleaves the best site within this region. Cleavage frequency at sites which do not conform closely to the consensus is, therefore, highly context dependent. Models and possible biological implications of these findings are discussed.Sequence-specific endonucleases are crucial for the proper maintenance of DNA in cells; they are required for its replication, recombination, and repair; its defense against invading parasites; and its proper degradation during programmed cell death in differentiated organisms. Scrutiny reveals strong similarities in their mechanisms of action, suggesting that some may participate in more than one such event.The restriction system encoded by coliphage T4 and dependent on its endonuclease II (EndoII) resembles type II restriction endonucleases more than type I or III or the methylationdependent endonucleases. Restriction enzymes recognize their specific DNA targets by a combination of direct and indirect readout (30, 31), i.e., interactions between nucleotide bases and amino acid residues and interactions between amino acids and the sugar-phosphate backbone, respectively. Both interactions are dependent on the sequence of the recognition site. Our previous investigations of cleavage in vivo of unmodified T4 DNA (24) as well as of a small plasmid (pBR322 [7]) suggested that recognition and cleavage by EndoII were dependent on helical structure. The helical structure of the recognition site may be influenced by factors outside the consensus sequence and may, in turn, influence the tracking of a processive enzyme along the DNA molecule.However, several features of T4 EndoII ...

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SummaryIn vivo , endonuclease II (EndoII) of coliphage T4 cleaves sites with conserved sequence elements (CSEs) to both the left and the right of the cleaved bonds, 16 bp altogether with some variability tolerated. In vitro , however, single-strand nicks in the lower strand predominate at sites containing only the left-side CSE that determines the precise position of lower strand nicks. Upper strand nick positions vary both in vivo and in vitro . A 24 bp substrate was nicked with the same precision as in longer substrates, showing that the conserved sequence suffices for precise nicking by EndoII. Using DNA ligase in vitro , we found that EndoII nicked both strands simultaneously at an in vivo -favoured site but not at an in vitro -favoured site. This indicates that the right-side CSE at in vivo -favoured sites is important for simultaneous nicking of both strands, generating doublestrand cleavage. Separate analysis of the two strands following in vitro digestion at two in vitro-favoured sites showed that EndoII nicked the lower strand about 1.5-fold faster than the upper strand. In addition, the upper and lower strands were nicked independently of each other, seldom resulting in doublestrand cleavage. Thus, cleavage by EndoII is the fortuitous outcome of two separate nicking events.