double-stranded restriction cleavage at 12 sites in pBR322 commence before 10-min postinfection with T4 at 37°C and proceeds more slowly in the presence of competing phage DNA than in its absence, utilizing the same sites in both cases. In a 200-base pair segment of the plasmid, single-stranded nicks also are frequent. The plasmid sites are cleaved with a speed that varies with the site, yielding frequencies of cleavage at different sites varying between 10 and 90%, at 50-min postinfection. All sites contain good matches to a consensus, 5'-GRCCGCNTYGC-3', most frequently cleaved around the variable central base pair, generating fragments with blunt ends or 1-2-base 5' overhangs. A larger consensus sequence, 5'-CGRCCGCNTTGSYNGC-3', has been identified. DNA sequence elements 3' to the cut site appear important for rapid cleavage
the phage T4 enzyme is involved in degradation of host DNA. The enzyme primarily catalyses nicking of the bottom strand of double stranded DNA between the first and second base pair to the right of a top-strand CCGC motif. Double-stranded breaks are produced 5fold to 10fold less frequently. It does not cleave the T4 native DNA, which contains 5-hydroxymethylcytosine instead of cytosine
EndoII nicks both strands simultaneously at an in vivo-favoured site but not at an in vitro-favoured site. The right-side conserved sequence element at in vivo-favoured sites is important for simultaneous nicking of both strands, generating double-strand cleavage. EndoII nicks the lower strand about 1.5fold faster than the upper strand. In addition, the upper and lower strands are nicked independently of each other, seldom resulting in double-strand cleavage
identification of seven major endonuclease II-dependent restriction sites in the T4 genome, in addition, abundant sites are cleaved in 55% of all molecules. Sites I, 11, and I11 share the sequence 5''CCGNNTTGGC-3' and are cleaved inabout 25% for I and III and 65% for II of all molecules, predominantly staggered around the first or second of the central unspecified base pairs to yield fragments with one 5' base. The less frequently cleaved sites I and I11 deviate from site I1 in predicted helical structure when viewed from the consensus strand, and in sequence when viewed from the opposite strand
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modeling based on strucuture of UvrC, PDB code 1YD0. Residues G49, R57, E118, and N130 in the putative catalytic surface are important for substrate recognition and binding. Residues G49 and R57 are essential for normal sequence recognition. and play a role in forming the DNA-binding surface and exposing the substrate scissile bond at the active site. Residues N130 and P127 likely contribute to positioning the catalytic domain correctly. Residue K76, part of a conserved NUMOD3 DNA-binding motif of homing endonucleases found to overlap the MR, affects both sequence recognition and catalysis
mutant E118A,crystallized in space group P21 with four monomers in the asymmetric unit. EndoII forms a striking X-shaped tetrameric structure composed as a dimer of dimers, with a protruding hairpin domain providing most of the dimerization and tetramerization interfaces. A bound phosphate ion in one of the four active sites of EndoII likely mimics the scissile phosphate in a true substrate complex. A protruding loop containing a nuclease-associated modular domain 3 element is likely to be involved in substrate binding, as well as residues forming a separate nucleic acid binding surface adjacent to the active site. The substrate would bind to a primary EndoII dimer diagonally over the active sites, requiring significant distortion of the enzyme or the substrate DNA, or both, for simultaneous nicking of both DNA strands. EndoII may bind its substrate inefficiently across the two sites in the dimer, offering a plausible explanation for the catalytic preponderance of single-strand nicks
Bacteriophage T4 endonucleases II and IV, oppositely affected by dCMP hydroxymethylase activity, have different roles in the degradation and in the RNA polymerase-dependent replication of T4 cytosine-containing DNA