Metabolic cofactors act as initiating substrates for primase and affect replication primer processing

Recently a new, non-canonical type of 5-RNA capping with cellular metabolic cofactors was discovered in bacteria and eukaryotes. This type of capping is performed by RNA polymerases, the main enzymes of transcription, which initiate RNA synthesis with cofactors. Here we show that primase, the enzyme of replication which primes synthesis of DNA by making short RNA primers, initiates synthesis of replication primers using the number of metabolic cofactors. Primase DnaG of E. coli starts synthesis of RNA with cofactors NAD+/NADH, FAD and DP-CoA in vitro. This activity does not affect primase specificity of initiation. ppGpp, the global starvation response regulator, strongly inhibits the non-canonical initiation by DnaG. Amino acid residues of a \"basic ridge\" define the binding determinant of cofactors to DnaG. Likewise, the human primase catalytic subunit P49 can use modified substrate m7GTP for synthesis initiation.\n\nFor correct genome duplication, the RNA primer needs to be removed and Okazaki fragments ligated. We show that the efficiency of primer processing by DNA polymerase I is strongly affected by cofactors on the 5-end of RNA. Overall our results suggest that cofactors at the 5 position of the primer influence regulation of initiation and Okazaki fragments processing.\n\nVisual abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY A. Non-canonical capping of RNA by RNA polymerase. RNA polymerase uses cellular cofactor as initiating substrate for RNA synthesis, instead of NTP. Then RNA chain grows, while cofactor remains attached and serves as cap. B. Proposed mechanism of non-canonical initiation of RNA primer synthesis by DnaG primase during replication. DnaG primase initiates synthesis of the primer for DNA replication using cellular cofactor. Primer stays annealed with the DNA template. DNApolI encounters cofactor, which affects the removal of primer.\n\nC_FIG_DISPLAY