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

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.

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

10.1101/741967 ◽

2019 ◽

Author(s):

Christina Julius ◽

Yulia Yuzenkova

Keyword(s):

Rna Polymerase ◽

Rna Synthesis ◽

Response Regulator ◽

Amino Acid Residues ◽

Okazaki Fragments ◽

Cellular Cofactor ◽

Canonical Type ◽

Rna Capping ◽

Polymerase I

AbstractRecently 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.For 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.Visual abstractA. 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.

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Escherichia coli β-clamp slows down DNA polymerase I dependent nick translation while accelerating ligation

10.1101/256537 ◽

2018 ◽

Author(s):

Amit Bhardwaj ◽

Debarghya Ghose ◽

Krishan Gopal Thakur ◽

Dipak Dutta

Keyword(s):

Escherichia Coli ◽

Dna Polymerase ◽

Nick Translation ◽

Dna Polymerase I ◽

Okazaki Fragments ◽

Pol I ◽

Dna Strands ◽

Translation Property ◽

Polymerase I

AbstractThe nick translation property of DNA polymerase I (Pol I) ensures the maturation of Okazaki fragments by removing primer RNAs and facilitating ligation. However, prolonged nick translation traversing downstream DNA is an energy wasting futile process, as Pol I simultaneously polymerizes and depolymerizes at the nick sites utilizing energy-rich dNTPs. Using an in vitro assay system, we demonstrate that the β-clamp of the Escherichia coli replisome strongly inhibits nick translation on the DNA substrate. To do so, β-clamp inhibits the strand displacement activity of Pol I by interfering with the interaction between the finger subdomain of Pol I and the downstream primer-template junction. Conversely, β-clamp stimulates the 5’ exonuclease property of Pol I to cleave single nucleotides or shorter oligonucleotide flaps. This single nucleotide flap removal at high frequency increases the probability of ligation between the upstream and downstream DNA strands at an early phase, terminating nick translation. Besides β-clamp-mediated ligation helps DNA ligase to seal the nick promptly during the maturation of Okazaki fragments.

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RNase HIII Is Important for Okazaki Fragment Processing inBacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.00686-18 ◽

2019 ◽

Vol 201 (7) ◽

Cited By ~ 4

Author(s):

Justin R. Randall ◽

Taylor M. Nye ◽

Katherine J. Wozniak ◽

Lyle A. Simmons

Keyword(s):

Dna Polymerase ◽

Dna Polymerase I ◽

Okazaki Fragment ◽

Okazaki Fragments ◽

Okazaki Fragment Processing ◽

Rnase Hi ◽

Okazaki Fragment Maturation ◽

Polymerase I

ABSTRACTRNA-DNA hybrids are common in chromosomal DNA. Persistent RNA-DNA hybrids result in replication fork stress, DNA breaks, and neurological disorders in humans. During replication, Okazaki fragment synthesis relies on frequent RNA primer placement, providing one of the most prominent forms of covalent RNA-DNA strandsin vivo. The mechanism of Okazaki fragment maturation, which involves RNA removal and subsequent DNA replacement, in bacteria lacking RNase HI remains unclear. In this work, we reconstituted repair of a linear model Okazaki fragmentin vitrousing purified recombinant enzymes fromBacillus subtilis. We showed that RNase HII and HIII are capable of incision on Okazaki fragmentsin vitroand that both enzymes show mild stimulation by single-stranded DNA binding protein (SSB). We also showed that RNase HIII and DNA polymerase I provide the primary pathway for Okazaki fragment maturationin vitro. Furthermore, we found that YpcP is a 5′ to 3′ nuclease that can act on a wide variety of RNA- and DNA-containing substrates and exhibits preference for degrading RNA in model Okazaki fragments. Together, our data showed that RNase HIII and DNA polymerase I provide the primary pathway for Okazaki fragment maturation, whereas YpcP also contributes to the removal of RNA from an Okazaki fragmentin vitro.IMPORTANCEAll cells are required to resolve the different types of RNA-DNA hybrids that formin vivo. When RNA-DNA hybrids persist, cells experience an increase in mutation rate and problems with DNA replication. Okazaki fragment synthesis on the lagging strand requires an RNA primer to begin synthesis of each fragment. The mechanism of RNA removal from Okazaki fragments remains unknown in bacteria that lack RNase HI. We examined Okazaki fragment processingin vitroand found that RNase HIII in conjunction with DNA polymerase I represent the most efficient repair pathway. We also assessed the contribution of YpcP and found that YpcP is a 5′ to 3′ exonuclease that prefers RNA substrates with activity on Okazaki and flap substratesin vitro.

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