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Structures of pMV158 replication initiator RepB with and without DNA reveal a flexible dual-function protein

Cristina Machón1,2, José A Ruiz-Masó3, Juliana Amodio1,2

  • 1Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology (BIST), Baldiri Reixac 10-12, 08028 Barcelona, Spain.

Nucleic Acids Research
|January 23, 2023
PubMed

Insights

Researchers revealed how the RepB protein initiates DNA replication on the pMV158 plasmid. Structural analysis shows specific elements enable RepB to bind DNA and cleave a strand, ensuring genetic fidelity.

Area of Science:

  • Molecular Biology
  • Genetics
  • Structural Biology

Background:

  • DNA replication ensures genetic material fidelity for cell division.
  • Rolling circle replication is a simple mechanism for replication initiation.
  • The streptococcal plasmid pMV158 utilizes RepB protein for replication initiation, conferring tetracycline resistance.

Purpose of the Study:

  • To elucidate the mechanism by which the RepB protein's origin binding domain recognizes and binds to the DNA bind locus.
  • To understand the structural basis of RepB's DNA cleavage activity at the nic locus.
  • To present a novel hexameric structure of full-length RepB and discuss its functional implications.

Main Methods:

  • Biochemical analyses were employed to study protein-DNA interactions.
  • Crystallographic analysis provided high-resolution structural insights into RepB.
  • Structural determination of a hexameric form of full-length RepB.

Main Results:

  • The RepB N-terminal domain utilizes a recognition α helix and a β-strand that organizes upon binding to recognize the DNA bind locus.
  • These structural elements are distinct from the active site, facilitating specific binding.
  • A hexameric structure of full-length RepB revealed significant protein flexibility, potentially enabling multiple functions.

Conclusions:

  • The study provides a detailed structural understanding of RepB's origin binding and DNA cleavage mechanism.
  • The flexibility of RepB, as shown in its hexameric structure, is crucial for its diverse roles in plasmid replication.
  • This work contributes to understanding the fundamental processes of DNA replication initiation in bacteria.

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