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Metabolic cofactors NADH and FAD act as non-canonical initiating substrates for a primase and affect replication primer processing in vitro.

Lookup NU author(s): Dr Christina Julius, Professor Paula SalgadoORCiD, Dr Yulia Yuzenkova

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This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).

Abstract

To initiate replication on a double-stranded DNA de novo, all organisms require primase, an RNA polymerase making short RNA primers which are then extended by DNA polymerases. Here, we show that primase can use metabolic cofactors as initiating substrates, instead of its canonical substrate ATP. DnaG primase of Escherichia coli initiates synthesis of RNA with NADH (the reduced form of nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide) in vitro. These cofactors consist of an ADP core covalently bound to extra moieties. The ADP component of these metabolites base-pairs with the DNA template and provides a 3'-OH group for RNA extension. The additional cofactors moieties apparently contact the 'basic ridge' domain of DnaG, but not the DNA template base at the -1 position. ppGpp, the starvation response regulator, strongly inhibits the initiation with cofactors, hypothetically due to competition for overlapping binding sites. Efficient RNA primer processing is a prerequisite for Okazaki fragments maturation, and we find that the efficiency of primer processing by DNA polymerase I in vitro is specifically affected by the cofactors on its 5'-end. Together these results indicate that utilization of cofactors as substrates by primase may influence regulation of replication initiation and Okazaki fragments processing.

Publication metadata

Author(s): Julius C, Salgado PS, Yuzenkova Y

Publication type: Article

Publication status: Published

Journal: Nucleic Acid Research

Year: 2020

Volume: 48

Issue: 13

Pages: 7298-7306

Print publication date: 27/11/2020

Online publication date: 28/05/2020

Acceptance date: 14/05/2020

Date deposited: 18/08/2021

ISSN (print): 0305-1048

ISSN (electronic): 1362-4962

Publisher: Oxford University Press

URL: https://doi.org/10.1093/nar/gkaa447

DOI: 10.1093/nar/gkaa447

PubMed id: 32463447

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