A comparison of eubacterial and archaeal structure-specific 5 '-exonucleases

The 8'-exonuclease domains of the DNA polymerase I proteins of Eubacteria a nd the FEN1 proteins of Eukarya and Archaea are members of a family of stru cture-specific 5'-exonucleases with similar function but limited sequence s imilarity. Their physiological role is to remove the displaced 5' strands c reated by DNA polymerase during displacement synthesis, thereby creating a substrate for DNA ligase, In this paper, we define the substrate requiremen ts for the 5'-exonuclease enzymes from Thermus aquaticus, Thermus thermophi lus, Archaeoglobus fulgidus, Pyrococcus furiosus, Methanococcus jannaschii, and Methanobacterium thermoautotrophicum, The optimal substrate of these e nzymes resembles DNA undergoing strand displacement synthesis and consists of a bifurcated downstream duplex with a directly abutted upstream duplex t hat overlaps the downstream duplex by one base pair. That single base of ov erlap causes the enzymes to leave a nick after cleavage and to cleave sever al orders of magnitude faster than a substrate that lacks overlap. The down stream duplex needs to be 10 base pairs long or greater for most of the enz ymes to cut efficiently. The upstream duplex needs to be only 2 or 3 base p airs long for most enzymes, and there appears to be interaction with the la st base of the primer strand. Overall, the enzymes display very similar sub strate specificities, despite their limited level of sequence similarity.