Calf 5' to 3' exo/endonuclease must slide from a 5' end of the substrate to perform structure-specific cleavage.
Calf 5' to 3' exo/endonuclease, the counterpart of the human FEN-1 and yeast RTH-1 nucleases, performs structure-specific cleavage of both RNA and DNA and is implicated in Okazaki fragment processing and DNA repair. The substrate for endonuclease activity is a primer annealed to a template but with a 5' unannealed tail. The results presented here demonstrate that the nuclease must enter the 5' end of the unannealed tail and then slide to the region of hybridization where the cleavage occurs. The presence of bound protein or a primer at any point on the single-stranded tail prevents cleavage. However, biotinylation of a nucleotide at the 5' end or internal to the tail does not prevent cleavage. The sliding process is bidirectional. If the nuclease slides onto the tail, later binding of a primer to the tail traps the nuclease between the primer binding site and the cleavage site, preventing the nuclease from departing from the 5' end. A model for 5' entry, sliding, and cleavage is presented. The possible role of this unusual mechanism in Okazaki fragment processing, DNA repair, and protection of the replication fork from inappropriate endonucleolytic cleavage is presented.
Department of Biochemistry, University of Rochester School of Medicine and Dentistry, New York 14642, USA.
R S
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Murante
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Rust
R A
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Bambara
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D004265:DNA Helicases / Q000378:metabolism; D017931:DNA Primers; D004260:DNA Repair; D004261:DNA Replication; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D045585:Flap Endonucleases; D006801:Humans; D007700:Kinetics; D008961:Models, Structural; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013379:Substrate Specificity; D013698:Templates, Genetic
D017931:DNA Primers; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases; D045585:Flap Endonucleases; C468317:FEN1 protein, human; D043211:Exodeoxyribonuclease V; D004265:DNA Helicases