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DNA replication is initiated at sites containing predefined DNA sequences known as origins of replication. DNA is unwound at these sites by the minichromosome maintenance (MCM) helicase and other factors such as Cdc45 and the associated GINS complex.The unwound single strands are protected by replication protein A (RPA) until DNA polymerase starts synthesizing DNA at the 5’ end of the strand in the same direction as the replication fork. To prevent the replication fork from falling apart,...
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In response to DNA damage, cells can pause the cell cycle to assess and repair the breaks. However, the cell must check the DNA at certain critical stages during the cell cycle. If the cell cycle pauses before DNA replication, the cells will contain twice the amount of DNA. On the other hand, if cells arrest after DNA replication but before mitosis, they will contain four times the normal amount of DNA. With a host of specialized proteins at their disposal,cells must use the right protein at...
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In response to DNA damage, cells can pause the cell cycle to assess and repair the breaks. However, the cell must check the DNA at certain critical stages during the cell cycle. If the cell cycle pauses before DNA replication, the cells will contain twice the amount of DNA. On the other hand, if cells arrest after DNA replication but before mitosis, they will contain four times the normal amount of DNA. With a host of specialized proteins at their disposal,cells must use the right protein at...
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Cdc45-induced loading of human RPA onto single-stranded DNA.

Anna Szambowska1, Ingrid Tessmer2, Piotr Prus3

  • 1Research Group Biochemistry, Leibniz Institute on Aging-Fritz Lipmann Institute, Beutenbergstrasse 11, D-07745 Jena, Germany.

Nucleic Acids Research
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Summary
This summary is machine-generated.

Cell division cycle protein 45 (Cdc45) facilitates DNA replication by loading RPA onto single-stranded DNA. This Cdc45-mediated process ensures orderly RPA binding at the replication fork.

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Area of Science:

  • Molecular Biology
  • DNA Replication
  • Protein-DNA Interactions

Background:

  • Cell division cycle protein 45 (Cdc45) is a key component of the eukaryotic replicative DNA helicase.
  • Understanding the precise role of Cdc45 in DNA replication is crucial for comprehending genome stability.

Purpose of the Study:

  • To investigate the interaction between human Cdc45 and the single-stranded DNA (ssDNA) binding protein RPA.
  • To elucidate the mechanism by which Cdc45 facilitates RPA loading onto nascent ssDNA during replication.

Main Methods:

  • Pull-down assays to study protein complex formation.
  • Surface plasmon resonance (SPR) to analyze binding kinetics.
  • Real-time analysis of RPA-ssDNA binding dynamics.

Main Results:

  • Human Cdc45 forms a complex with RPA and actively loads it onto ssDNA.
  • Cdc45-bound RPA binds ssDNA in an 8-10 nucleotide mode, dissociating at 30 nucleotides.
  • Cdc45-mediated RPA loading is catalytic and requires physical contact with the RPA70A subdomain.

Conclusions:

  • Cdc45 controls RPA binding modes on ssDNA, stabilizing the 8-nt mode and facilitating transition to the 30-mer mode.
  • Cdc45 ensures ordered RPA deposition on newly synthesized ssDNA at the replication fork.
  • This Cdc45-mediated loading mechanism is essential for seamless DNA replication.