title: Cleavage of double-stranded DNA by the intrinsic 3'-5' exonuclease activity of DNA polymerase B1 from the hyperthermophilic archaeon Sulfolobus solfataricus at high temperature.
abstract: The substrate requirement of the intrinsic 3'-5' exonuclease of DNA polymerase B1 from the hyperthermophilic archaeon Sulfolobus solfataricus P2 (Sso polB1) was investigated. Sso polB1 degraded both single-stranded (ss) and double-stranded (ds) DNA at similar rates in vitro at temperatures of physiological relevance. No difference was found in the cleavage of 3'-recessive, 3'-protruding and blunt-ended DNA duplexes at these temperatures. However, a single-stranded nick in duplex DNA was less readily employed by the enzyme to initiate cleavage than a free 3' end. At lower temperatures, Sso polB1 cleaved ssDNA more efficiently than dsDNA. The strong 3'-5' exonuclease activity of polB1 was inhibited by 50% in the presence of 2 microM dNTPs, but remained measurable at up to 600 microM dNTPs. In view of the strong exonuclease activity of Sso polB1 on matched dsDNA, we suggest that S. solfataricus may have evolved mechanisms to regulate the exonuclease/polymerase ratio of the enzyme, thereby reducing the cost of proofreading at high temperature.
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, PR China.
authors.forename: Huiqiang
authors.initials: H
authors.lastname: Lou
authors.forename: Zhenhong
authors.initials: Z
authors.lastname: Duan
authors.forename: Tong
authors.initials: T
authors.lastname: Sun
authors.forename: Li
authors.initials: L
authors.lastname: Huang
mesh_terms: D004247:DNA / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D005092:Exonucleases / Q000378:metabolism*; D007700:Kinetics; D016944:Sulfolobus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D013696:Temperature
chemical_list: D004247:DNA; D004259:DNA-Directed DNA Polymerase; D005092:Exonucleases