An Autoinhibited Conformation of the DnaB-Replicative Helicase – phage λP Loader Complex
Abstract Replicative helicases are closed protein rings and require loader proteins for assembly on DNA at origins of DNA replication. Multiple copies of the bacterial DnaC or the phage λP loader proteins bind to the closed planar ring of the DnaB replicative helicase and trigger its reconfiguration into an open spiral conformation wherein an internal chamber becomes accessible for entry to physiologically produced single stranded DNA at the replication origin. Although a great deal was learned from a previously determined cryo-EM structure of theE. coliDnaB helicase bound to the phage helicase loader λP (BP), the pentameric λP ensemble was not well resolved and this stymied deeper insights. We revised have the BP structure using X-ray and AlphaFold determined structures to interpret a 2.8Å cryo-EM density map. We find that the λP ensemble adopts a profoundly asymmetric configuration; one copy of λP, which is visualized in full, binds at the top and bottom of the open DnaB spiral; presence of a single breach in the DnaB open spiral means that the remaining four copies of λP must adopt distinct, and currently unknown, conformations. Although DnaB’s internal chamber remains accessible to entry of ssDNA, the λP protomer whose binding site spans the breach effectively blocks the path into the inner chamber and gives rise to an autoinhibited configuration for the BP complex. Comparisons of the λP and DnaC bound complexes of the DnaB helicase shed new light on how the two loaders, though unrelated in sequence or structure, converged on the same ring-opening mechanism. The autoinhibited conformation of the BP complex suggests structural changes that must accompany recruitment to the initiator protein complex at the replication origin..
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Preprint| Erscheinungsjahr: |
2023 |
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| Erschienen: |
2023 |
| Enthalten in: |
bioRxiv.org - (2023) vom: 03. Jan. Zur Gesamtaufnahme - year:2023 |
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| Sprache: |
Englisch |
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| Beteiligte Personen: |
Brown, Dhanjai [VerfasserIn] |
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| Links: |
Volltext [kostenfrei] |
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| doi: |
10.1101/2022.12.30.522210 |
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| PPN (Katalog-ID): |
XBI038292963 |
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| 245 | 1 | 0 | |a An Autoinhibited Conformation of the DnaB-Replicative Helicase – phage λP Loader Complex |
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| 520 | |a Abstract Replicative helicases are closed protein rings and require loader proteins for assembly on DNA at origins of DNA replication. Multiple copies of the bacterial DnaC or the phage λP loader proteins bind to the closed planar ring of the DnaB replicative helicase and trigger its reconfiguration into an open spiral conformation wherein an internal chamber becomes accessible for entry to physiologically produced single stranded DNA at the replication origin. Although a great deal was learned from a previously determined cryo-EM structure of theE. coliDnaB helicase bound to the phage helicase loader λP (BP), the pentameric λP ensemble was not well resolved and this stymied deeper insights. We revised have the BP structure using X-ray and AlphaFold determined structures to interpret a 2.8Å cryo-EM density map. We find that the λP ensemble adopts a profoundly asymmetric configuration; one copy of λP, which is visualized in full, binds at the top and bottom of the open DnaB spiral; presence of a single breach in the DnaB open spiral means that the remaining four copies of λP must adopt distinct, and currently unknown, conformations. Although DnaB’s internal chamber remains accessible to entry of ssDNA, the λP protomer whose binding site spans the breach effectively blocks the path into the inner chamber and gives rise to an autoinhibited configuration for the BP complex. Comparisons of the λP and DnaC bound complexes of the DnaB helicase shed new light on how the two loaders, though unrelated in sequence or structure, converged on the same ring-opening mechanism. The autoinhibited conformation of the BP complex suggests structural changes that must accompany recruitment to the initiator protein complex at the replication origin. | ||
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| 650 | 4 | |a 570 |7 (dpeaa)DE-84 | |
| 700 | 1 | |a Shatarupa, Abhipsa |e verfasserin |4 aut | |
| 700 | 1 | |a Olinares, Paul Dominic B. |e verfasserin |4 aut | |
| 700 | 1 | |a Chase, Jillian |e verfasserin |4 aut | |
| 700 | 1 | |a Isiorho, Eta |e verfasserin |4 aut | |
| 700 | 1 | |a Chait, Brian T. |e verfasserin |4 aut | |
| 700 | 1 | |a Jeruzalmi, David |e verfasserin |4 aut | |
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