The DNA primase of Sulfolobus solfataricus is activated by substrates containing a thymine-rich bubble and has a 3'-terminal nucleotidyl-transferase activity.
DNA primases are responsible for the synthesis of the short RNA primers that are used by the replicative DNA polymerases to initiate DNA synthesis on the leading- and lagging-strand at the replication fork. In this study, we report the purification and biochemical characterization of a DNA primase (Sso DNA primase) from the thermoacidophilic crenarchaeon Sulfolobus solfataricus. The Sso DNA primase is a heterodimer composed of two subunits of 36 kDa (small subunit) and 38 kDa (large subunit), which show sequence similarity to the eukaryotic DNA primase p60 and p50 subunits, respectively. The two polypeptides were co-expressed in Escherichia coli and purified as a heterodimeric complex, with a Stokes radius of about 39.2 A and a 1:1 stoichiometric ratio among its subunits. The Sso DNA primase utilizes poly-pyrimidine single-stranded DNA templates with low efficiency for de novo synthesis of RNA primers, whereas its synthetic function is specifically activated by thymine-containing synthetic bubble structures that mimic early replication intermediates. Interestingly, the Sso DNA primase complex is endowed with a terminal nucleotidyl-transferase activity, being able to incorporate nucleotides at the 3' end of synthetic oligonucleotides in a non-templated manner.
De Falco
Mariarosaria
M
Istituto di Biochimica delle Proteine, Consiglio Nazionale delle Ricerche, Via P. Castellino, 111, 80131-Napoli, Italy.
Fusco
Alessandra
A
De Felice
Mariarita
M
Rossi
Mosè
M
Pisani
Francesca M
FM
D000255:Adenosine Triphosphate / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D019915:DNA Primase / Q000302:isolation & purification / Q000378:metabolism*; D004789:Enzyme Activation; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D009713:Nucleotidyltransferases / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D009843:Oligoribonucleotides / Q000096:biosynthesis / Q000737:chemistry; D011071:Poly T / Q000737:chemistry* / Q000378:metabolism; D012313:RNA / Q000096:biosynthesis; D013379:Substrate Specificity; D016944:Sulfolobus / Q000201:enzymology*; D013698:Templates, Genetic
D009838:Oligodeoxyribonucleotides; D009843:Oligoribonucleotides; C034477:RNA primers; D011071:Poly T; D012313:RNA; D000255:Adenosine Triphosphate; D004247:DNA; D019915:DNA Primase; D009713:Nucleotidyltransferases