The DNA-stimulated ATPase characterized in the accompanying paper is shown to be a DNA unwinding enzyme. Substrates employed were DNA . RNA hybrid duplexes and D N A . DNA partial duplexes prepared by polymerization on fd phage single-stranded DNA template. The enzyme was found to denature these duplexes in an ATP-dependent reaction, without detectably degrading. EDTA, an inhibitor of the Mg2 +-requiring ATPase, was found to prevent denaturation suggesting that dephosphorylation of the ATP and not only its presence is required. These results together with those from enzyme-DNA binding studies lead to ideas regarding the mode of enzymic action. It is proposed that the enzyme binds, in an initial step, to a single-stranded part of the DNA substrate molecule and that from here, energetically supported by ATP dephosphorylation, it invades double-stranded parts separating base-paired strands by processive, zipper-like action. It is further proposed that chain separation results from the combined action of several enzyme molecules and that a tendency of the enzyme to aggregate with itself reflects a tendency of the molecules to cooperate. Various functions are conceivable for the enzyme.In the preceding paper [l] we have described a novel DNA-stimulated ATPase purified from Escherichiu coli. The enzyme, an MgZ +-requiring y-phosphohydrolase, has a peptide molecular weight of approximately 180000 and exists, in high salt, as a monomer of probably highly anisometric shape. In low salt, where the ATPase activity is highest, the enzyme forms aggregates. The enzyme binds, as the purification procedures shows, to single-stranded DNA which also best satisfies its cofactor requirement and does not detectably cleave DNA.The present publication shows that the ATPase activity is used for denaturing base-paired DNA. Abbreviation. EGTA, ethylene glycol-bis(2-aminoethylether) N,N'-tetradcetate. MATERIALS AND METHODS BuffersEnzymes. DNA-stimulated ATPdse (EC 3.6.1.3); DNA polymerase I (EC 2.7.7.7); RNA polymerase (EC 2.7.7.6); S1 nuclease (EC 3.1.4.21).cerol, 100 pg bovine serum albumin/ml. Buffer 11 is 20 mM Tris/maleate buffer (pH 7.2), 2.5 mM MgClZ, 15 mM 2-mercaptoethanol, 10 % glycerol. EnzymesDNA unwinding ATPase, fraction 5, was prepared as described in the accompanying paper. E. coli DNA polymerase I and RNA polymerase were gifts from Dr K . Geider. S1 nuclease from Aspergillus oryzae was purified through the DEAE-cellulose step PI. Nucfeic AcidsDNA preparations were those used in the accompanying paper. 3H-labelled @X DNA was a gift of Dr U. Hess.DNA . RNA hybrid duplexes were prepared according to Wickner et ul. [3] by transcribing on 16 nmol (as nucleotide) fd DNA with 20 pg RNA polymerase in the presence of three non-radioactive ribonucleoside triphosphates (250 pM) and [5-3H]-CTP (New England) (1.1 pM; 23.2 Ci/mmol) in 1.6 ml final volume. After 20 min at 30 "C the reaction was interrupted with 5 mM EDTA and 1 % sodium dodecylsulphate. The mixture (4.6 x lo6 acid-precipitable 3H counts/min) was then extracted with phen...

Ein fädiger DNS-Phage (fd) und ein sphärischer RNS-Phage (fr), wirtsspezifisch für männliche Stämme von E. coli 1. P rä p a ra tio n und chemische Eigenschaften von fd und fr Von H a r t m u t H o f f m a n n -Be r l i n g , D o n a l d A. M a r v i n und H i l d e g a r d D ü r w a l d Bacteriophage fd has the physical form of a flexible rod. It contains 12.2% DNA of m.w. 1.37x10®, calculated from the particle weight of the phage (Part II). Base ratios and optical behavior indicate that the DNA is single-stranded. Thymine (34.1%) predominates as is found for other single-stranded DNA phages. Because of its composition and high yield, the phage delivers large amounts of coat protein.Bacteriophage fr contains 30% RNA of m.w. 1.2 x 10®, calculated from the molecular weight of the phage (Part I I ). The RNA, after extraction from the coat protein, forms a more ordered structure.Unpurified preparations of the phages fd and fr contain particles -probably incomplete phageswhich are made up of the specific phage coat protein and a reduced proportion of nucleic acid.Convenient methods for the concentration and purification of the phages are described.Die Coli-Phagen 1 7 4 1 und f 2 2 verkörpern eine neue Gruppe von Bakteriophagen, die durch ihre Kleinheit und O rganisation von den klassischen Phagen verschieden sind. Die kleinen Phagen enthal ten als kennzeichnende Besonderheit Nucleinsäure in der Form eines Einzelstrangs, der bei X aus D N S 3, bei f2 aus RNS gebildet ist. f2 hat einen eigenartigen W irtsbereich, der die Auffindung ähn licher RNS-haltiger Phagen in aller Welt erleichtert hat ® a' W ir berichten über zwei weitere Phagen mit der für f2 kennzeichnenden W irtsspezifität, fd und fr *. fr enthält RNS und ist den beschriebenen RNS-Phagen ähnlich; fd enthält DNS, besitzt die für Phagen ungewöhnliche Form eines Stäbchens und unter scheidet sich von allen bekannten Phagen dadurch, daß fd nicht durch Lyse der infizierten Bakterien freigesetzt wird (vgl. Teil III). fd und fr entstam men der gleichen Abwässerprobe, haben den glei chen, eng begrenzten W irtsbereich und eine schwache serologische Ähnlichkeit. Die Phagen differieren durch ihre Gestalt, die chemische N atur ihrer Nucleinsäuren und die verschiedenen Proportionen der Nucleobasen. 1 R. L. S i k s h e i m e r , J. molecular Biol. 1, 37 [1959]. 2 T. L o e b u . N. D. Z i n d e r , Proc. nat. Acad. Sei. USA 47. 282 u. 742 [1961]. 3 R. L. Si n s h e i m e r . Medien Tryptone-Medium ist ähnlich dem Medium von L o e b und Z i n d e r 2 und enthält pro l 10 g Difco-Tryptone, 1 g Difco-Yeast-Extract, 2,5 g NaCl, 2 -10~3-m. CaCl2 und als Puffer 1 0 -2-m. Imidazol ph 7,2. Festes Medium wird ohne Imidazol und mit einem Zusatz von 1 % Agar hergestellt. Imidazolhaltige Medien können vollständig zusammengesetzt autoklaviert werden. Pufferlösung enthält (in M o l//): NaCl 0,15; Trispuffer pn 7,6 0,02; M gCl, 10~3; Gelatine 2 -IO-3 Gramm. Vermehrung und Reinigung der Phagen 1. fd fd wurde auf E. coli K 12 W 945 (F + (/.) -pili , von Dr. C. C. B r i n t o n ) vermehrt. ...

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The DNA-stimulated 75000-M, ATPase described in the preceding paper is shown to be a further catalytic DNA unwinding principle (DNA unwinding enzyme 11) made in Escherichia coli cells (the first being the 180000-M, ATPase of the cells: DNA unwinding enzyme I). Unwinding depends, strictly, on the supply of ATP. It occurs only under conditions permitting ATP dephosphorylation and it proceeds as long as enzyme molecules are permitted to enter the enzyme . DNA complex. The enzyme binds specifically to single-stranded DNA yielding a complex of only limited stability. These results are interpreted in terms of a distributive mode of action of the enzyme. It is argued that chain separation starts near a single-stranded DNA region and that, forced by continued adsorption of enzyme molecules to the DNA, it develops along the duplex. This mechanism is different from that deduced previously for DNA unwinding enzyme I. Complicated results were obtained using ATPase prepared from rep3 mutant cells.The DNA-dependent ATP-y-phosphohydrolase described in the preceding paper [l] is a globular molecule of M , 75000 purified from Escherichia coli. The ATPase is specific for nucleoside triphosphates containing adenine and it prefers single-stranded DNA as the cofactor. Attempts at purifying the ATPase from a variety of mutant strains showed that the yield is unusually variable when rep3 mutant cells (unable to propagate bacteriophage 4 X 174 DNA [2]) are used for extraction.In this paper we will show that the ATPase activity can be exploited for unwinding duplex DNA. As proposed in the preceding paper we will call the previously described, fibrous, 180 000-M, DNA unwinding ATPase of E. coli the DNA unwinding enzyme I of this organism and the 75000-M, ATPase the DNA unwinding enzyme 11.

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