The 5-exonuclease domains of the DNA polymerase I proteins of Eubacteria and the FEN1 proteins of Eukarya and Archaea are members of a family of structurespecific 5-exonucleases with similar function but limited sequence similarity. Their physiological role is to remove the displaced 5 strands created by DNA polymerase during displacement synthesis, thereby creating a substrate for DNA ligase. In this paper, we define the substrate requirements for the 5-exonuclease enzymes from Thermus aquaticus, Thermus thermophilus, Archaeoglobus fulgidus, Pyrococcus furiosus, Methanococcus jannaschii, and Methanobacterium thermoautotrophicum. The optimal substrate of these enzymes resembles DNA undergoing strand displacement synthesis and consists of a bifurcated downstream duplex with a directly abutted upstream duplex that overlaps the downstream duplex by one base pair. That single base of overlap causes the enzymes to leave a nick after cleavage and to cleave several orders of magnitude faster than a substrate that lacks overlap. The downstream duplex needs to be 10 base pairs long or greater for most of the enzymes to cut efficiently. The upstream duplex needs to be only 2 or 3 base pairs long for most enzymes, and there appears to be interaction with the last base of the primer strand. Overall, the enzymes display very similar substrate specificities, despite their limited level of sequence similarity.The 5Ј nuclease domains of DNA polymerase I from Escherichia coli and Thermus aquaticus were the first extensively characterized members of a large class of structure-specific 5Ј-exonucleases (1, 2). Initially it was proposed that these enzymes work as true exonucleases removing predominantly mono-or dinucleotides from the 5Ј end of double-stranded DNA (3). More detailed studies have shown that 5Ј nucleases of this type specifically recognize bifurcated ends of double-stranded regions and remove single-stranded 5Ј arms by cutting the phosphodiester bond after the first base pair of the duplex, leaving a 3Ј hydroxyl end (2). A mammalian enzyme with functional similarity to the 5Ј-exonuclease domain of E. coli polymerase I was isolated nearly 30 years ago (4). Later, additional members of this group of enzymes called flap endonucleases (FEN1) from Eukarya and Archaea were shown to possess a nearly identical structure-specific activity (5-8), although they have limited sequence similarity to the bacterial 5Ј-exonuclease proteins.The substrate specificities of the FEN1 enzymes and the eubacterial and related bacteriophage enzymes have been examined and found to be similar for all enzymes (2, 5, 6, 8 -11). The minimal requirement for cleavage is a bifurcated duplex with a free 5Ј end. The presence of an upstream primer that directly abuts the downstream strand stimulates cleavage, but its precise effect on the site of cleavage remains unclear. In the majority of studies that were done with the flap substrate described in Harrington et al. (5), the enzymes leave predominately a 1-nucleotide gap or 1-nucleotide overlap between t...

The invasive signal amplification reaction is a sensitive method for single nucleotide polymorphism detection and quantitative determination of viral load and gene expression. The method requires the adjacent binding of upstream and downstream oligonucleotides to a target nucleic acid (either DNA or RNA) to form a specific substrate for the structure-specific 5' nucleases that cleave the downstream oligonucleotide to generate signal. By running the reaction at an elevated temperature, the downstream oligonucleotide cycles on and off the target leading to multiple cleavage events per target molecule without temperature cycling. We have examined the performanceof the FEN1 enzymes from Archaeoglobus fulgidus and Methanococcus jannaschii and the DNA polymerase I homologues from Thermus aquaticus and Thermus thermophilus in the invasive signal amplification reaction. We find that the reaction has a distinct temperature optimum which increases with increasing length of the downstream oligonucleotide. Raising the concentration of either the downstream oligonucleotide or the enzyme increases the reaction rate. When the reaction is configured to cycle the upstream instead of the downstream oligonucleotide, only the FEN1 enzymes can support a high level of cleavage. To investigate the origin of the background signal generated during the invasive reaction, the cleavage rates for several nonspecific substrates that arise during the course of a reaction were measured and compared with the rate of the specific reaction. We find that the different 5' nuclease enzymes display a much greater variability in cleavage rates on the nonspecific substrates than on the specific substrate. The experimental data are compared with a theoretical model of the invasive signal amplification reaction.

We describe here a new approach for analyzing nucleic acid sequences using a structure-specific endonuclease, Cleavase I. We have applied this technique to the detection and localization of mutations associated with isoniazid resistance in Mycobacterium tuberculosis and for differentiating bacterial genera, species, and strains. The technique described here is based on the observation that single strands of DNAs can assume defined conformations, which can be detected and cleaved by structure-specific endonucleases such as Cleavase I. The patterns of fragments produced are characteristic of the sequences responsible for the structures, so that each DNA has its own structural fingerprint. Amplicons containing either a single 5-fluorescein or 5-tetramethyl rhodamine label were generated from a 620-bp segment of the katG gene of isoniazid-resistant and-sensitive M. tuberculosis, the 5 350 bp of the 16S rRNA genes of Escherichia coli O157:H7, Salmonella typhimurium, Salmonella enteritidis, Salmonella arizonae, Shigella sonnei, Shigella dysenteriae, Campylobacter jejuni, Staphylococcus hominis, Staphylococcus warneri, and Staphylococcus aureus and an approximately 550-bp DNA segment comprising the intergenic region between the 16S and 23S rRNA genes of Salmonella typhimurium, Salmonella enteritidis, Salmonella arizonae, Shigella sonnei, and Shigella dysenteriae serotypes 1, 2, and 8. Changes in the structural fingerprints of DNA fragments derived from the katG genes of isoniazid-resistant M. tuberculosis isolates were clearly identified and could be mapped to the site of the actual mutation relative to the labeled end. Band patterns which clearly differentiated bacteria to the level of genus and, in some cases, species were generated from the 16S genes. Cleavase I analysis of the intergenic regions of Salmonella and Shigella species differentiated genus, species, and serotypes. Structural fingerprinting by digestion with Cleavase I is a rapid, simple, and sensitive method for analyzing nucleic acid sequences and may find wide utility in microbial analysis.