An endonuclease was detected in strains ofSalmonella typhimurium containing the drug resistance plasmid pKM101. The enzyme was not detectable in strains lacking this plasmid, but it was present in strains containing mutants of pKM101 that were no longer able to enhance host cell mutagenesis. The endonuclease had a molecular weight of roughly 75,000 and, at pH 7.0, was equally active on single-stranded and duplex deoxyribonucleic acid (DNA). The reaction with single-stranded DNA was optimal at pH 5.5, whereas with duplex DNA the optimum was pH 6.8. The enzyme required a divalent cation for activity, and it had no detectable exonuclease activity with single-stranded or duplex DNA. The endonuclease extensively degraded DNA with no apparent base specificity, forming 5'-phosphomonoester termini. Although characterization ofthe endonuclease has not revealed its function, the enzyme does not appear to be a restriction endonuclease.

Methyl groups placed on 4XsB1 replicative form DNA DNA, and since all observed mutations leading to loss of the site occur at one of the bases specified by this sequence. Analysis of the sequence of gXam3cs70 showed that if no other residues are recognized, all seven of these bases are essential for recognition and the interval between the two groups of specified bases must be precisely eight.The restriction enzyme found in Escherichia coil B is classified as a type I restriction endonuclease because it requires ATP and S-adenosylmethionine (AdoMet) for enzymatic activity (1, 2). Because the enzyme does not cleave DNA at unique sites (2, 3) the DNA sequence recognized by the enzyme could not be determined by analysis of the termini of its cleavage products. However, the enzyme must recognize specific nucleotides prior to the cleavage step, since if the DNA is "modified" by a specific methylase activity, it no longer is susceptible to the endonuclease (2). The methyl groups placed on simian virus 40 (SV40) (4) or phage fi replicative form (RF) DNA (5) by this activity have been localized to small restriction fragments. Furthermore, mutants of phages fd (6) and fi (7) exist that have lost susceptibility to restriction by E. coil B and a mutant of cX174 has been isolated that has gained this susceptibility (8). The sites of the mutations of fl have been mapped to specific regions of the genome.The sequences recognized by several restriction enzymes have been determined by mapping sites cleaved by these enzymes within tracts of DNA whose sequences have been determined and examining such sequences for homology (1).

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A novel form of DNA polymerase I (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, DNA nucleotidyltransferase, EC 2.7.7.7) activity has been isolated from Escherichia coli cells that had been activated for expression of the DNA damage-inducible genes. Induction was by treatment ofnormal cells or cells carrying the #pr-51 and tif-1 mutations with nalidixic acid. This activity, DNA polymerase I*, seems to be a form ofDNA polymerase I because it is insensitive toN-ethylmaleimide, is inhibited by antibody to DNA polymerase I, and does not appear in a polAl strain. DNA polymerase I* activity sediments through sucrose gradients as a broad peak with s2 ,0 = 6.6-10.5, compared with an s2o,. = 4.8-5.5 for DNA polymerase I. The fidelity during polymerization reactions of DNA polymerase I* is relatively low with a variety of synthetic templates and deoxynucleoside triphosphates, although the enzyme appears to have a normal level of 3'->5' exonuclease. Polymerase I* has properties that might implicate it in some form of mutagenic DNA repair.Mutations induced in Escherichia coli by UV radiation or by a variety of chemicals are believed to often be the consequence of an error-prone DNA repair pathway controlled, along with other SOS functions, by the recA and lexA genes (1-3). This pathway is evidently induced by agents that block DNA replication either by interfering with replicative enzymes or by modifying bases so as to affect base recognition in the DNA template. Blocks due to base damage are presumably mediated by constraints that regulate replicative fidelity by ensuring the synthesis only of properly base-paired DNA. Models have been proposed for overcoming such replicative blocks that predict the transient appearance of error-prone forms of DNA polymerase that can polymerize random nucleotides opposite template damage (2,4). Because such hypotheses are consistent with much of the available data on mutagenic DNA repair (5), we have sought to identify these predicted forms of DNA polymerase. We report here the observation ofan error-prone form of DNA polymerase I that is associated with the induction of recA/lexA functions in E. coli. MATERIALS AND METHODSBacterial Strains. Extracts were prepared from E. coli K-12 strains DM1187 (tif-1, spr-51, lexA3, sftAll, his4, strA31) (6), AB1157 (argE3, his4, leu-6, proA2, thr-1, ara-14, galK2, lacYl, mtll, xyl5, thi-l, tsx-33, supE44, sup-37, str-31) (7), and P3478 (polAl, thyA36), a derivative of W3110 (5).Materials. Synthetic polymers and unlabeled dNTPs were from P-L Biochemicals, radioactive nucleotides were from Amersham, and electrophoretically homogeneous DNA polymerase I was a gift from Arthur Kornberg, Stanford University, or Lawrence Loeb, University ofWashington. Antibody against DNA polymerase I was provided by I. R. Lehman, Stanford University. Polymin-P was from Miles; agarose (type II) was from Sigma; DNA-agarose was prepared by the method of Schaller et aL (8), except that DNA had been denatured by heating to 950C.Sucrose Gradient Sedimenta...

The purification is reported of an endopeptidase, XSCEPl (Xenopus skin cysteine endopeptidase), present in skin secretions of Xenopus. The procedure involved an initial concentration of the enzyme by batchwise anionexchange chromatography and ammonium sulphate precipitation. The proteolytic activity, determined with ZPhe-Arg-Amc (Z, benzyloxycarbonyl; Amc, 7-amidomethylcoumarin) as substrate, was fractionated by gradient ion-exchange chromatography, yielding a major component which was purified to homogeneity by chromatography on an organomercury-agarose column. SDSjPAGE demonstrated the presence of a single protein with a molecular mass of 27 kDa. The purified enzyme, which possesed a pH optimum of 5.5, exhibited the properties of a cysteine endopeptidase: it was activated by dithiothreitol and EDTA and inhibited by the mechanism-based inhibitor trans-epoxysuccinyl-~-leucylamido(4-guanidino)butane. XSCEPl exhibited a marked preference for substrates with a hydrophobic residue in the PI position and arginine in the P2 position as opposed to a substrate with arginine residues in both positions. The enzyme was also able to cleave a Val-Arg-Gly sequence in a model substrate, reflecting cleavages undergone by a number of peptides present in Xenopus skin. The results point to a functional role for XSCEPl as a putative processing enzyme.The skin glands of Xenopus laevis secrete a wide range of bioactive peptides, many of which exhibit sequence homology with mammalian peptide hormones and neurotransmitters [l, 21. Although the precise biological function of the skin peptides is not known, several exhibit anti-microbial and membranolytic activity by virtue of their ability to form membrane-disrupting helices [3 -51. The secreted peptides arise from pro-peptides in processing reactions that in many intances are analogous to the reactions undergone by their mammalian counterparts [4, 61. Processing generally commences with cleavage of the pro-peptide at single or paired basic residue sites [7], the basic residues are removed from the resulting fragments by the action of an exopeptidase [8] and peptides terminating in glycine undergo C-terminal amidationThe skin peptides are stored in secretory vesicles within the epidermal gland until release is triggered by adrenergic stimulus [lo]. After secretion, the peptides are cleaved by an endopeptidase which acts N-terminally to lysine residues, either inactivating the peptide or possibly generating a product with a new biological activity [Ill.Recently it has been reported that an amidating enzyme [I21 and a dipeptidyl aminopeptidase [I31 which appear to be involved in pro-peptide processing are secreted together with Xenopus skin peptides and, notably, there are marked similarities in the sequence and properties of the amidating enzyme ~91.Correspondence to D. G. Smyth with mammalian amidating enzymes [9, 141. Furthermore comparison of sequences around certain single basic residue cleavage sites in Xenopus and mammalian pro-peptides suggests that similar structural features di...