A comparison of eubacterial and archaeal structure-specific 5'-exonucleases.
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 structure-specific 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.
Kaiser
M W
MW
Third Wave Technologies, Inc., Madison, Wisconsin 53719, USA. mkaiser@twt.com
Lyamicheva
N
N
Ma
W
W
Miller
C
C
Neri
B
B
Fors
L
L
Lyamichev
V I
VI
D001105:Archaea / Q000201:enzymology*; D001419:Bacteria / Q000201:enzymology*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004256:DNA Polymerase I / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D043264:Phosphodiesterase I; D010727:Phosphoric Diester Hydrolases / Q000235:genetics / Q000378:metabolism*; D017421:Sequence Analysis; D013379:Substrate Specificity
D001426:Bacterial Proteins; D004256:DNA Polymerase I; D010727:Phosphoric Diester Hydrolases; D043264:Phosphodiesterase I