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Replication origin-flanking roadblocks reveal origin-licensing dynamics and altered sequence dependence.

Megan D Warner1, Ishara F Azmi1, Sukhyun Kang1

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The Journal of Biological Chemistry
|October 28, 2017
PubMed
Summary
This summary is machine-generated.

Researchers investigated DNA replication origin licensing in eukaryotes. They found that a sliding intermediate, not precise DNA binding, dictates Mcm2-7 helicase loading, allowing for more flexible origin recognition.

Keywords:
ATPases associated with diverse cellular activities (AAA)DNA helicaseDNA replicationDNA–protein interactionSaccharomyces cerevisiaecdt1cell cyclehelicase loadingnucleosomeorigin of replication

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Eukaryotic DNA replication requires timely genome duplication initiated from multiple origins.
  • Origin licensing involves loading the Mcm2-7 helicase complex onto DNA, typically mediated by ORC and Cdc6 proteins.
  • Current models propose ORC and Cdc6 encircle origin DNA to assemble the Mcm2-7 double-hexamer.

Purpose of the Study:

  • To test the precise location of Mcm2-7 helicase loading during origin licensing.
  • To investigate the role of DNA-protein interactions and sliding intermediates in this process.
  • To assess the influence of roadblocks and nucleosomes on origin recognition and helicase loading.

Main Methods:

  • Introduction of DNA-protein roadblocks (HpaII methyltransferase or TALE protein) adjacent to the ORC-binding site in *Saccharomyces cerevisiae* origin DNA.
  • Analysis of Mcm2-7 helicase loading defects in the presence of these roadblocks.
  • Evaluation of ORC-DNA binding and helicase loading under various roadblock configurations.

Main Results:

  • Helicase loading defects were observed only with roadblocks that inhibited ORC-DNA binding.
  • Inhibition of helicase loading without affecting ORC-DNA binding occurred when roadblocks flanked the origin, restricting a sliding intermediate.
  • Eliminating one flanking roadblock removed the loading defect, supporting a sliding mechanism.
  • Origin-flanking nucleosomes or roadblocks induced dependence on a weaker ORC-DNA-binding site for helicase loading.

Conclusions:

  • The precise DNA sequence and binding site are not the sole determinants of Mcm2-7 helicase loading.
  • A sliding helicase-loading intermediate model explains the observed flexibility in origin licensing.
  • This sliding mechanism accommodates variations in DNA sequence requirements for origin activation.