Primer initiation and extension by T7 DNA primase.
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.
Qimron
Udi
U
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
Lee
Seung-Joo
SJ
Hamdan
Samir M
SM
Richardson
Charles C
CC
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
D017931:DNA Primers; D021122:Protein Subunits; D012265:Ribonucleotides; D019915:DNA Primase