title: Primer initiation and extension by T7 DNA primase.
abstract: T7 DNA primase is composed of a catalytic RNA polymerase domain (RPD) and a zinc-binding domain (ZBD) connected by an unstructured linker. The two domains are required to initiate the synthesis of the diribonucleotide pppAC and its extension into a functional primer pppACCC (de novo synthesis), as well as for the extension of exogenous AC diribonucleotides into an ACCC primer (extension synthesis). To explore the mechanism underlying the RPD and ZBD interactions, we have changed the length of the linker between them. Wild-type T7 DNA primase is 10-fold superior in de novo synthesis compared to T7 DNA primase having a shorter linker. However, the primase having the shorter linker exhibits a two-fold enhancement in its extension synthesis. T7 DNA primase does not catalyze extension synthesis by a ZBD of one subunit acting on a RPD of an adjacent subunit (trans mode), whereas de novo synthesis is feasible in this mode. We propose a mechanism for primer initiation and extension based on these findings.
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Udi
authors.initials: U
authors.lastname: Qimron
authors.forename: Seung-Joo
authors.initials: SJ
authors.lastname: Lee
authors.forename: Samir M
authors.initials: SM
authors.lastname: Hamdan
authors.forename: Charles C
authors.initials: CC
authors.lastname: Richardson
mesh_terms: D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D019915:DNA Primase / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D012265:Ribonucleotides / Q000235:genetics / Q000378:metabolism*; D013698:Templates, Genetic; D014158:Transcription, Genetic
chemical_list: D017931:DNA Primers; D021122:Protein Subunits; D012265:Ribonucleotides; D019915:DNA Primase