Structural analyses of the bacterial primosomal protein DnaB reveal that it is a tetramer and forms a complex with a primosomal re-initiation protein: Bacterial DnaB: a loader for PriA mediated DNA replication

Yi-Ching Li, Vankadari Naveen, Min-Guan Lin, Chwan-Deng Hsiao

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5 Citations (Scopus)

Abstract

The DnaB primosomal protein from Gram-positive bacteria plays a key role in DNA replication and restart as a loader protein for the recruitment of replisome cascade proteins. Previous investigations have established that DnaB is composed of an N-terminal domain, a middle domain, and a C-terminal domain. However, structural evidence for how DnaB functions at the atomic level is lacking. Here, we report the crystal structure of DnaB, encompassing the N-terminal and middle domains (residues 1-300), from Geobacillus stearothermophilus (GstDnaB1-300) at 2.8 Å resolution. Our structure revealed that GstDnaB1-300 forms a tetramer with two basket-like architecture, a finding s consistent with those from solution studies using analytical ultracentrifugation. Furthermore, our results from both GST pull-down assays and analytical ultracentrifugation show that GstDnaB1-300 is sufficient to form a complex with PriA, the primosomal re-initiation protein. Moreover, with the aid of small angle X-ray scattering (SAXS) experiments, we also determined the structural envelope of full-length DnaB (GstDnaBFL) in solution. These SAXS studies indicated that GstDnaBFL has an elongated conformation and that the protruding density envelopes originating from GstDnaB1-300 could completely accommodate the GstDnaB C-terminal domain (residues 301-461) . Taken together with biochemical assays, our results suggest that GstDnaB uses different domains to distinguish the PriA-interaction and ssDNA-binding. This finding can further extend our understanding of primosomal assembly in replication restart.
Original languageEnglish
Pages (from-to)15744-15757
Number of pages14
JournalThe Journal of Biological Chemistry
Volume292
Issue number38
DOIs
Publication statusPublished - 22 Sep 2017
Externally publishedYes

Keywords

  • DNA-protein interaction
  • gram-positive bacteria
  • protein-protein interaction
  • small-angle X-ray scattering (SAXS)
  • X-ray crystallography
  • helicase loader protein
  • primosome assembly
  • replication restart

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