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

Christina Julius,Paula S Salgado,Yulia Yuzenkova

doi:10.1093/nar/gkaa447

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.

Highlights

This work was inspired by the recent discovery of the non-canonical RNA capping phenomenon
We wanted to test if primase can initiate synthesis using ADPcontaining metabolic cofactors, by analogy with other polymerases
We found that DnaG makes a 13nt long RNA product in a subset of NTPs using either ATP, NAD+, NADH, FAD and DP-coA (Fig. 1B)

Summary

Introduction

This work was inspired by the recent discovery of the non-canonical RNA capping phenomenon. Many RNA species in bacteria and eukaryotes bear metabolic adenine-containing cofactors at their 5’-end; NAD+ (nicotinamide adenine dinucleotide) and DP-CoA (dephospho- coenzyme A) and FAD (flavin adenine dinucleotide), as well as cell wall precursors UDP-GlcNAc (uridine 5’-diphospho-N-acetylglucosamine) and UDP-Glc (uridine 5’-diphosphate glucose) [1, 2]. Unlike the classic cap m7G, noncanonical caps are installed by the main enzyme of transcription, RNA polymerase (RNAP)(3). It happens during initiation of transcription in a template-dependent manner – ADP-containing cofactors are incorporated at promoters with +1A start sites, i.e. promoters dictating ATP as the initiating substrate [3, 4], UDP-containing cell wall precursors - on +1U promoters [3]. NudC processes NADylated RNAs into a monophosphorylated species that are quickly degraded in the cell [5]

Methods

Results

Conclusion