A mobile genetic element-derived primase-polymerase harbors multiple activities implicated in DNA replication and...

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Primase-polymerases (PrimPols) are essential enzymes involved in DNA replication and repair across all domains of life.
• Diverse PrimPols encoded by mobile genetic elements (MGEs) function as replicases, but their activities remain incompletely understood.

Purpose of the Study:

• To characterize primase-polymerases genetically associated with prokaryotic argonaute proteins (pAgos).
• To elucidate the dual roles of pAgo-associated PrimPol (AgaPP) in DNA replication and repair processes.

Main Methods:

• Biochemical characterization of AgaPP's polymerase and primase activities.
• Investigation of AgaPP's interaction with a co-encoded helicase.
• Assessing AgaPP's role in translesion DNA synthesis, terminal transfer, and microhomology-mediated end joining (MMEJ).
• Evaluating AgaPP's efficacy in repairing Cas9-induced DNA breaks and enhancing cell survival in Escherichia coli.

Main Results:

• AgaPP exhibits polymerase and primase activities and forms a functional replication module with a partner helicase.
• AgaPP demonstrates translesion synthesis, terminal transfer, and MMEJ capabilities, similar to human DNA repair polymerase θ.
• AgaPP facilitates MMEJ repair of double-stranded DNA breaks, improving E. coli survival after DNA damage.
• The MMEJ activity of AgaPP is applicable for in vitro DNA assembly.

Conclusions:

• AgaPP, likely originating from an MGE, possesses a dual function in both DNA replication and repair.
• AgaPP represents a novel DNA repair polymerase with potential applications in biotechnology and synthetic biology.