Structural Insight into the DNA-Binding Mode of the Primosomal Proteins PriA, PriB, and DnaT

Huang, Yen‐Hua; Huang, Cheng-Yang

doi:10.1155/2014/195162

Cited by 25 publications

(32 citation statements)

References 103 publications

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“…In one, the duplex has been modeled to interact with the OB-fold, through some contacts used to bind single strands plus others. In another model, a different binding site mimics binding of double-stranded DNA by the E. coli histone-like HU protein but still shares some interactions with the OB fold (30). Our findings could be explained by either model, provided novel contacts occur that are closer to the ends of the protein than the OB-fold single-stranded DNA contacts.…”

Section: Discussionmentioning

confidence: 71%

“…Binding takes place through an OB fold in a slightly different mode that uses different interactions (16,30). Unlike SSB, PriB binds both single-and double-stranded DNA (31).…”

Section: Discussionmentioning

confidence: 99%

“…It is possible that the binding observed near the distal end of the duplex arises from fraying, because no DNA-DNA cross-link is observed. No structure is available for PriB binding doublestranded DNA, but two models have been proposed (30). In one, the duplex has been modeled to interact with the OB-fold, through some contacts used to bind single strands plus others.…”

Section: Discussionmentioning

confidence: 99%

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Identification of Subunit Binding Positions on a Model Fork and Displacements That Occur during Sequential Assembly of the Escherichia coli Primosome

Manhart

McHenry

2015

Journal of Biological Chemistry

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Background: Photo-cross-linking revealed the position of replication restart protein interactions with a fork. Results: PriA cross-links to the fork until DnaC, DnaB, and ATP␥S are added. Conclusion: PriA remains stably associated with forks until the final stage of helicase assembly, when it is displaced. Significance: The approach employed should be applicable to complex protein-nucleic acid assembly reactions.

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Section: Discussionmentioning

confidence: 71%

“…Binding takes place through an OB fold in a slightly different mode that uses different interactions (16,30). Unlike SSB, PriB binds both single-and double-stranded DNA (31).…”

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Identification of Subunit Binding Positions on a Model Fork and Displacements That Occur during Sequential Assembly of the Escherichia coli Primosome

Manhart

McHenry

2015

Journal of Biological Chemistry

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“…NS3 helicase was also overlaid, although not bound to a similar ssDNA-dsDNA structure, because its putative strand-separation pin has been identified (28,33). As has been noted (13,24,34), the overlay showed that a ␤-hairpin from the PriA CRR (residues 452-462 in E. coli PriA (Fig. 1A)) was located at a similar position to pins in other helicases, implicating it as a putative strand-separation pin.…”

Section: Pria-dna Fork Cross-linking Maps the Crr ␤-Hairpin To The Lomentioning

confidence: 85%

Function of a strand-separation pin element in the PriA DNA replication restart helicase

Windgassen

Leroux

Sandler

et al. 2019

Journal of Biological Chemistry

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Edited by Patrick Sung DNA helicases are motor proteins that couple the chemical energy of nucleoside triphosphate hydrolysis to the mechanical functions required for DNA unwinding. Studies of several helicases have identified strand-separating "pin" structures that are positioned to intercept incoming dsDNA and promote strand separation during helicase translocation. However, pin structures vary among helicases and it remains unclear whether they confer a conserved unwinding mechanism. Here, we tested the biochemical and cellular roles of a putative pin element within the Escherichia coli PriA DNA helicase. PriA orchestrates replication restart in bacteria by unwinding the lagging-strand arm of abandoned DNA replication forks and reloading the replicative helicase with the help of protein partners that combine with PriA to form what is referred to as a primosome complex. Using in vitro protein-DNA cross-linking, we localized the putative pin (a ␤-hairpin within a zinc-binding domain in PriA) near the ssDNA-dsDNA junction of the lagging strand in a PriA-DNA replication fork complex. Removal of residues at the tip of the ␤-hairpin eliminated PriA DNA unwinding, interaction with the primosome protein PriB, and cellular function. We isolated a spontaneous intragenic suppressor mutant of the priA ␤-hairpin deletion mutant in which 22 codons around the deletion site were duplicated. This suppressor variant and an Ala-substituted ␤-hairpin PriA variant displayed wildtype levels of DNA unwinding and PriB binding in vitro. These results suggest essential but sequence nonspecific roles for the PriA pin element and coupling of PriA DNA unwinding to its interaction with PriB. Figure 6. The CRR ␤-hairpin serves a role in PriB interaction with the PriA-DNA complex. Native PAGE of EMSA of the synthetic DNA fork (1 nM; depicted to left of gel; used in Fig. 4 except nascent leading strand was included and nascent lagging strand excluded) incubated with PriA variant (2 nM) and increasing concentrations of PriB (0, 10, 40, and 160 nM monomers). First lane is DNA fork alone. Second lane is PriB (160 nM) without PriA. Representative gel of three replicates.

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“…The mechanisms of action of replication restart primosome in the Gram-negative Escherichia coli have been established [9][10][11]. In E. coli, the replication restart primosome consists of PriA helicase, PriB, PriC, DnaB helicase, DnaC, DnaT, and DnaG primase [12]. In the Gram-positive Bacillus subtilis, the DNA replication initiator protein PriA helicase has homolog of E. coli [13].…”

Section: Introductionmentioning

confidence: 99%

Inhibition of Staphylococcus aureus PriA Helicase by Flavonol Kaempferol

Huang

Chen

et al. 2015

Protein J

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Staphylococcus aureus is an important etiological agent responsible for healthcare-associated infections. In this study, the effect of flavonoids on the inhibition of S. aureus PriA (SaPriA), an essential helicase for DNA replication restart, which is critical for bacterial survival, was investigated. Using vanadate-sensitive colorimetric assay, the concentration of phosphate, from ATP hydrolysis by SaPriA, was decreased to 37 and 69%, respectively, in the presence of 35 μM kaempferol and myricetin. The effect of quercetin, galangin, dihydromyricetin, and myricitrin was insignificant. From titration curve, IC50 of kaempferol for SaPriA was determined to be 22 ± 2 μM. Using fluorescence quenching, we identified that kaempferol can bind to SaPriA with K(d) of 9.1 ± 3.2 μM. To our knowledge, these preliminary results constituted the first study regarding that naturally occurring product such as flavonols kaempferol and myricetin can be potent inhibitors targeting PriA.

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Structural Insight into the DNA-Binding Mode of the Primosomal Proteins PriA, PriB, and DnaT

Cited by 25 publications

References 103 publications

Identification of Subunit Binding Positions on a Model Fork and Displacements That Occur during Sequential Assembly of the Escherichia coli Primosome

Identification of Subunit Binding Positions on a Model Fork and Displacements That Occur during Sequential Assembly of the Escherichia coli Primosome

Function of a strand-separation pin element in the PriA DNA replication restart helicase

Inhibition of Staphylococcus aureus PriA Helicase by Flavonol Kaempferol

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