The heterodimeric primase of the hyperthermophilic archaeon Sulfolobus solfataricus possesses DNA and RNA primase, polymerase and 3'-terminal nucleotidyl transferase activities.
A eukaryotic-type primase was identified in the crenarchaeon Sulfolobus solfataricus. The two-subunit DNA-dependent primase, termed PriSL, was purified following co-expression of the subunits in Escherichia coli and its activity was characterised. PriSL was capable of utilising both ribonucleotides and deoxyribonucleotides for primer synthesis in the presence of natural, or synthetic, single-stranded DNA. A broad distribution of products was detected, ranging from dinucleotides to DNA molecules in excess of 7 kb and RNA up to 1 kb in length. However, PriSL had a significantly higher affinity for ribonucleotides than for deoxyribonucleotides. Using site-directed mutagenesis, two aspartate residues crucial for nucleic acid synthesis and residues important for the binding of free nucleotides were identified. In addition to the primase and polymerase activities, we reveal that the primase possesses a template-independent 3'-terminal nucleotidyl transferase activity.
Lao-Sirieix
Si-houy
SH
MRC Cancer Cell Unit, Hutchison MRC Research Centre, Hills Road, Cambridge CB2 2XZ, UK.
Bell
Stephen D
SD
D000595:Amino Acid Sequence; D002384:Catalysis; D004247:DNA / Q000096:biosynthesis / Q000378:metabolism; D019915:DNA Primase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019281:Dimerization; D008958:Models, Molecular; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D009711:Nucleotides / Q000378:metabolism; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000096:biosynthesis / Q000378:metabolism*; D016415:Sequence Alignment; D048229:Sulfolobus solfataricus / Q000201:enzymology* / Q000235:genetics; D013696:Temperature
D046912:Multiprotein Complexes; D009711:Nucleotides; D021122:Protein Subunits; D012313:RNA; D004247:DNA; D019915:DNA Primase; D009713:Nucleotidyltransferases