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Related Experiment Videos

A DNA structure is required for geminivirus replication origin function

B M Orozco1, L Hanley-Bowdoin

  • 1Department of Biochemistry, North Carolina State University, Raleigh 27695-7622, USA.

Journal of Virology
|January 1, 1996
PubMed
Summary
This summary is machine-generated.

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The hairpin structure within the tomato golden mosaic virus (TGMV) origin of replication is crucial for viral DNA replication. Its sequence influences replication efficiency, highlighting its essential role in the viral life cycle.

Area of Science:

  • Plant Virology
  • Molecular Biology
  • DNA Replication

Background:

  • Geminiviruses, like tomato golden mosaic virus (TGMV), possess a genome of two single-stranded circular DNAs (A and B).
  • Replication occurs via a rolling-circle mechanism within the host plant cell nucleus.
  • The origin of replication contains essential elements, including a recognition site for the AL1 replication protein and a motif capable of forming hairpin/cruciform structures.

Purpose of the Study:

  • To investigate the functional significance of the hairpin motif at the TGMV origin of replication.
  • To determine how structural integrity and sequence variations of this motif impact viral replication.
  • To elucidate the role of the TGMV AL1 protein in initiating replication.

Main Methods:

  • Construction and analysis of TGMV B-component DNA mutants with altered hairpin structures.

Related Experiment Videos

  • Replication assays in tobacco protoplasts, both in transient assays with helper DNA and with viral replication proteins provided in trans.
  • In vitro site-specific endonuclease assays using the TGMV AL1 protein.

    • Main Results:

    • Mutant B DNAs retaining the hairpin structure replicated efficiently when AL1 protein was supplied.
    • Replication efficiency was significantly reduced in transient assays with replicating wild-type TGMV A DNA if the hairpin structure was compromised.
    • The TGMV AL1 protein functions as a site-specific endonuclease, cleaving DNA at the hairpin loop, with activity correlating to replication phenotypes.

    Conclusions:

    • The hairpin structure is essential for TGMV replication initiation.
    • The sequence of the hairpin motif modulates replication efficiency.
    • DNA cleavage by AL1 at the hairpin loop is a critical step in initiating rolling-circle replication.