Modulation of hyperthermophilic DNA polymerase activity by archaeal chromatin proteins.
Sulfolobus synthesizes a large quantity of highly conserved 7-kDa DNA-binding proteins suspected to be involved in chromosomal organization. The effect of the 7-kDa proteins on the polymerization and 3'-5' exonuclease activities of a family B DNA polymerase (polB1) from the hyperthermophilic archaeon Sulfolobus solfataricus was investigated. polB1 degraded both single-stranded DNA and double-stranded DNA at similar rates in vitro at temperatures of physiological relevance. The 7-kDa proteins were capable of significantly inhibiting the excision and enhancing the extension of matched template primers by the polymerase. However, the proteins did not protect single-stranded DNA from cleavage by polB1. In addition, the 7-kDa proteins did not affect the proofreading ability of polB1 and were not inhibitory to the excision of mismatched primers by the polymerase. The dNTP concentrations required for the effective inhibition of the 3'-5' exonuclease activity of polB1 were lowered from approximately 1 mm in the absence of the 7-kDa proteins to approximately 50 microm in the presence of the proteins at 65 degrees C. Our data suggest that the 7-kDa chromatin proteins serve to modulate the extension and excision activities of the hyperthermophilic DNA polymerase, reducing the cost of proofreading by the enzyme at high temperature.
Lou
Huiqiang
H
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, People's Republic of China.
Duan
Zhenhong
Z
Huo
Xiaofeng
X
Huang
Li
L
D019843:Archaeal Proteins / Q000378:metabolism*; D001483:Base Sequence; D002843:Chromatin / Q000378:metabolism*; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D005092:Exonucleases / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016944:Sulfolobus / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D013698:Templates, Genetic; D013816:Thermodynamics
D019843:Archaeal Proteins; D002843:Chromatin; D017931:DNA Primers; C072146:Pfu DNA polymerase; D004259:DNA-Directed DNA Polymerase; D005092:Exonucleases