Primer initiation and extension by T7 DNA primase

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• T7 DNA primase, crucial for DNA replication initiation, comprises an RNA polymerase domain (RPD) and a zinc-binding domain (ZBD).
• These domains collaborate in both de novo primer synthesis (pppAC to pppACCC) and extension synthesis using exogenous diribonucleotides.
• Understanding the interplay between RPD and ZBD is key to elucidating primase function.

Purpose of the Study:

• To investigate the functional consequences of modifying the linker connecting the RPD and ZBD in T7 DNA primase.
• To explore how linker length influences the distinct mechanisms of de novo and extension primer synthesis.
• To gain insights into the interaction dynamics between the RPD and ZBD domains during T7 primase activity.

Main Methods:

• Site-directed mutagenesis was employed to alter the linker length between the RPD and ZBD of T7 DNA primase.
• Enzymatic assays were performed to quantify the efficiency of de novo primer synthesis (pppAC to pppACCC).
• Extension synthesis assays were conducted using exogenous AC diribonucleotides to assess primer elongation.

Main Results:

• Wild-type T7 DNA primase demonstrated a 10-fold higher efficiency in de novo synthesis compared to primase with a shortened linker.
• Conversely, T7 DNA primase with a shortened linker exhibited a two-fold enhancement in extension synthesis efficiency.
• De novo synthesis was observed to occur in a trans mode (inter-subunit interaction), while extension synthesis did not utilize this mode.

Conclusions:

• Linker length critically modulates the functional specialization of T7 DNA primase domains.
• The findings suggest distinct mechanisms for de novo and extension synthesis, involving different RPD-ZBD interaction modes.
• A mechanistic model for T7 DNA primase primer initiation and extension is proposed based on linker length-dependent domain interactions.