A question remaining to be answered about RecA protein function concerns the role of ATP hydrolysis during the DNA-strand-exchange reaction. In this paper we describe the formation ofjoint molecules in the absence of ATP hydrolysis, using adenosine 5'-[r-thio]triphosphate (ATP[yS]) as nucleotide cofactor. Upon the addition of double-stranded DNA, the ATP[ySJ-RecA protein-single-stranded DNA presynaptic complexes can form homologously paired molecules that are stable after deproteinization. Formation of these joint molecules requires both homology and a free homologous end, suggesting that they are plectonemic in nature. This reaction is very sensitive to magnesium ion concentration, with a maximum rate and extent observed at 4-5 mM magnesium acetate. Under these conditions, the average length of heteroduplex DNA within the joint molecules is 2.4-3.4 kilobase pairs. Thus, RecA protein can form extensive regions of heteroduplex DNA in the presence of ATP [yS], suggesting that homologous pairing and the exchange of the DNA molecules can occur without ATP hydrolysis. A model for the RecA protein-catalyzed DNA-strand-exchange reaction that incorporates these results and its relevance to the mechanisms ofeukaryotic recombinases are presented.The RecA protein-catalyzed DNA-strand-exchange reaction has been divided into at least three distinct phases (1, 2). In the first phase of this reaction, RecA protein binds to single-stranded DNA (ssDNA) in the presence of ATP (presynapsis). During the second phase, synapsis, the ssDNA and double-stranded DNA (dsDNA) (4)]. Since plectonemic, as well as paranemic, joint molecules can be formed in the presence of ATP, it was suggested that conversion of paranemic to plectonemic joint molecules requires ATP hydrolysis (4). In related studies, addition ofATP[yS] to an ongoing DNA-strand-exchange reaction was shown to immediately stop formation of heteroduplex DNA (3). The data were interpreted to suggest that ATP hydrolysis was also required for the formation of extensive regions of heteroduplex DNA (after joint molecules are formed). Thus, homologous pairing and the formation of protein-stabilized paranemic joint molecules are thought to occur in the absence of ATP hydrolysis, whereas the formation of plectonemic joint molecules and extensive regions of heteroduplex DNA requires hydrolysis of ATP. We now report the catalysis ofDNA strand exchange in the presence of ATP [yS]. Our data demonstrate that the joint molecules formed in the presence ofATP [yS] are stable in the absence of RecA protein and are, therefore, plectonemic in nature. The average length of heteroduplex DNA in these joint molecules can be as much as 3.4 kilobase pairs (kb) with no detectable hydrolysis of ATP [yS]. The relevance of these results to the role of ATP hydrolysis in the mechanism of the DNA strand exchange catalyzed by the Escherichia coli RecA protein and by other recombinases is discussed. MATERIALS AND METHODSReagents. All chemicals used were reagent grade and solutions were made in gla...

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The DNA-dependent protein kinase (DNA-PK) is required for DNA double-strand break (DSB) repair and immunoglobulin gene rearrangement and may play a role in the regulation of transcription. The DNA-PK holoenzyme is composed of three polypeptide subunits: the DNA binding Ku70͞86 heterodimer and an Ϸ460-kDa catalytic subunit (DNA-PKcs). DNA-PK has been hypothesized to assemble at DNA DSBs and play structural as well as signal transduction roles in DSB repair. Recent advances in atomic force microscopy (AFM) have resulted in a technology capable of producing high resolution images of native protein and proteinnucleic acid complexes without staining or metal coating. The AFM provides a rapid and direct means of probing the protein-nucleic acid interactions responsible for DNA repair and genetic regulation. Here we have employed AFM as well as electron microscopy to visualize Ku and DNA-PK in association with DNA. A significant number of DNA molecules formed loops in the presence of Ku. DNA looping appeared to be sequence-independent and unaffected by the presence of DNA-PKcs. Gel filtration of Ku in the absence and the presence of DNA indicates that Ku does not form nonspecific aggregates. We conclude that, when bound to DNA, Ku is capable of self-association. These findings suggest that Ku binding at DNA DSBs will result in Ku self-association and a physical tethering of the broken DNA strands.

The antiviral activities of poly(phenylene ethynylene) (PPE)-based cationic conjugated polyelectrolytes (CPE) and oligo-phenylene ethynylenes (OPE) were investigated using two model viruses, the T4 and MS2 bacteriophages. Under UV/visible light irradiation, significant antiviral activity was observed for all of the CPEs and OPEs; without irradiation, most of these compounds exhibited high inactivation activity against the MS2 phage and moderate inactivation ability against the T4 phage. Transmission electron microscopy (TEM) and SDS polyacrylamide gel electrophoresis (SDS-PAGE) reveal that the CPEs and OPEs exert their antiviral activity by partial disassembly of the phage particle structure in the dark and photochemical damage of the phage capsid protein under UV/visible light irradiation.

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The Escherichia coli uvrD gene product, helicase II, is required for both methyl-directed mismatch and uvrABC excision repair and is believed to function by unwinding duplex DNA. Initiation of unwinding may occur specifically at either a mismatch or a nick, although no direct evidence for this has previously been reported. It has recently been shown that helicase II can unwind fully duplex linear and nicked circular DNA with lengths of at least =2700 base pairs in vitro; hence, a flanking region of single-stranded DNA is not required to initiate DNA unwinding. In studies with uniquely nicked duplex DNA, we present EM evidence that helicase II protein initiates DNA unwinding at the nick, with unwinding proceeding bidirectionally. We also show that helicase II protein initiates DNA unwinding at the blunt ends of linear DNA, rather than in internal regions. These data provide direct evidence that helicase II protein can initiate unwinding of duplex DNA at a nick, in the absence of auxiliary proteins. We propose that helicase II may initiate unwinding from a nick in a number of DNA repair processes.The Escherichia coli helicase II protein, the product of the uvrD gene (1-4), is a DNA-dependent ATPase and helicase (5, 6), which catalyzes the unwinding of duplex DNA with an apparent 3' to 5' polarity (7). Helicase II has been implicated to function in replication (8), as well as recombination (9-11). This protein is also required in methyl-directed mismatch (12-14) and uvrABC excision repair pathways (15-17), both of which require a nicked DNA as an intermediate. In methyl-directed mismatch repair, helicase II protein has been proposed to initiate DNA unwinding at the mismatch (18), although initiation at the nick has not been ruled out. Most previous studies of DNA unwinding by helicase II in vitro have concluded that a flanking region of single-stranded DNA (ssDNA), 3' to the duplex, is required to initiate unwinding (7,19 MATERIALS AND METHODSBuffers and Enzymes. Buffers were made with distilled deionized Milli-Q water or, for EM, two times glass-distilled water that was redistilled within 12 hr of use. TE buffer is 10 mM Tris-HCl (pH 8.1)/1 mM Na3EDTA. Unwinding buffer (pH 7.5 at 370C) is 40 mM Hepes'KOH/0.1 mM dithiothreitol/1.5 mM ATP/0.5 mM MgCl2.Helicase II protein was purified from E. coli N4830/ pTLS1, and its concentration was determined spectrophotometrically (20). Helicase II protein stocks were tested for exonuclease activity on both 5' and 3' end-labeled ss-and duplex DNA under the same conditions used in the unwinding experiments. Helicase II and 32P end-labeled DNA were incubated at 370C and then subjected to electrophoresis on polyacrylamide gels. After 45 min, <0.4% of the 32p label had been removed from the ends of the DNA substrates. fi gene II protein was purified from E. coli K561/pDG117IIA (21), according to S. Johnston and D. Ray (University of California at Los Angeles) with some modifications.DNA Substrates. M13mpll replicative form (RF) DNA and pUC8 DNA were isolated as described (...

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