Structural and mechanistic insights into primer synthesis initiation by DNA primase

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• DNA primases are essential enzymes for genome replication, synthesizing RNA or DNA primers.
• The precise mechanism of initial dinucleotide synthesis by primases remains largely unelucidated.
• Understanding primer initiation is critical for comprehending DNA replication fidelity.

Purpose of the Study:

• To elucidate the mechanism of initial dinucleotide formation by a minimal primase.
• To characterize the structural intermediates involved in primer synthesis initiation.
• To identify conserved features of primase function in primer formation.

Main Methods:

• Utilized nucleotide analogues to capture transient reaction intermediates.
• Employed Nuclear Magnetic Resonance (NMR) spectroscopy for structural analysis.
• Performed computational modeling to refine structural insights.

Main Results:

• The ancillary domain of the primase binds both the DNA template and initiating nucleotides.
• Only the second nucleotide base-pairs with the template; the first remains unpaired, inducing base flipping.
• A closed conformation positions nucleotides for catalysis, revealing template recognition and proofreading mechanisms.

Conclusions:

• A novel mechanism for DNA primer initiation involving template-base flipping and specific nucleotide interactions was uncovered.
• This mechanism facilitates nucleotide assembly and proofreading during the critical first step of primer synthesis.
• The findings suggest a conserved mechanism for primer initiation across diverse primases.