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Related Experiment Videos

The reconstituted human Chl12-RFC complex functions as a second PCNA loader.

Yasushi Shiomi1, Ayako Shinozaki, Katsunori Sugimoto

  • 1Department of Biology, School of Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.

Genes to Cells : Devoted to Molecular & Cellular Mechanisms
|April 7, 2004
PubMed
Summary

The human Chl12-RFC complex acts as a second proliferating cell nuclear antigen (PCNA) loader, distinct from replication factor C (RFC). This complex plays a unique role in DNA replication, facilitating PCNA loading and stimulating DNA synthesis.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Cell Biology

Background:

  • Chl12 is a sister chromatid cohesion factor with sequence similarity to RFC1.
  • Chl12 forms a clamp loader complex with RFC small subunits (RFCs2-5).

Purpose of the Study:

  • To investigate the function of the human Chl12-RFC complex.
  • To determine if the Chl12-RFC complex interacts with proliferating cell nuclear antigen (PCNA).
  • To elucidate the role of the Chl12-RFC complex in DNA replication.

Main Methods:

  • Reconstitution of the human Chl12-RFC complex using a baculovirus expression system.
  • Electron microscopy to assess structural similarity to RFC.
  • In vitro assays to measure DNA-stimulated ATPase activity, DNA binding, PCNA loading, and DNA polymerase delta activity.

Related Experiment Videos

  • Comparison with replication factor C (RFC) in simian virus 40 DNA replication assays.

    • Main Results:

    • The human Chl12-RFC complex specifically interacts with human PCNA.
    • Structurally, the Chl12-RFC complex is indistinguishable from RFC.
    • The complex exhibits DNA-stimulated ATPase activity, enhanced by PCNA and DNA binding.
    • Chl12-RFC loads PCNA onto DNA and stimulates DNA polymerase delta synthesis.
    • Chl12-RFC cannot substitute for RFC in simian virus 40 DNA replication.

    Conclusions:

    • The human Chl12-RFC complex functions as a second PCNA loader.
    • The roles of Chl12-RFC in DNA replication are distinct from those of RFC.