Distinct domain functions regulating de novo DNA synthesis of thermostable DNA primase from hyperthermophile Pyrococcus horikoshii.
DNA primases are essential components of the DNA replication apparatus in every organism. Reported here are the biochemical characteristics of a thermostable DNA primase from the thermophilic archaeon Pyrococcus horikoshii, which formed the oligomeric unit L(1)S(1) and synthesized long DNA primers 10 times more effectively than RNA primers. The N-terminal (25KL) and C-terminal halves (20KL) of the large subunit (L) play distinct roles in regulating de novo DNA synthesis of the small catalytic subunit (S). The 25KL domain has a dual function. One function is to depress the large affinity of the intrasubunit domain 20KL for the template DNA until complex (L(1)S(1) unit) formation. The other function is to tether the L subunit tightly to the S subunit, probably to promote effective interaction between the intrasubunit domain 20KL and the active center of the S subunit. The 20KL domain is a central factor to enhance the de novo DNA synthesis activity of the catalytic S subunit since the total affinity of the L(1)S(1) unit is mainly derived from the affinity of 20KL, which is elevated more than 10 times by the heterodimer formation, presumably due to the cancellation of the inhibitory activity of 25KL through tight binding to the S subunit.
Matsui
Eriko
E
Biological Information Research Center, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305-8566, Japan.
Nishio
Miho
M
Yokoyama
Hideshi
H
Harata
Kazuaki
K
Darnis
Sophie
S
Matsui
Ikuo
I
D000595:Amino Acid Sequence; D019843:Archaeal Proteins; D001483:Base Sequence; D011232:Chemical Precipitation; D019915:DNA Primase / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D004261:DNA Replication / Q000235:genetics; D019641:DNA, Archaeal / Q000096:biosynthesis* / Q000302:isolation & purification; D004795:Enzyme Stability / Q000235:genetics; D006639:Histidine / Q000737:chemistry; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D021122:Protein Subunits / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D043902:Pyrococcus horikoshii / Q000201:enzymology* / Q000235:genetics*; D019642:RNA, Archaeal / Q000096:biosynthesis; D020349:Surface Plasmon Resonance
D019843:Archaeal Proteins; D019641:DNA, Archaeal; D021122:Protein Subunits; D019642:RNA, Archaeal; D006639:Histidine; D019915:DNA Primase; C443135:DNA primase, Pyrococcus