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Functional sites of human PCNA which interact with p21 (Cip1/Waf1), DNA polymerase delta and replication factor C
1Faculty of Biological Science, Nara Institute of Science and Technology, Takayama, Ikoma, Japan.
Background:
PCNA, an eukaryotic DNA sliding clamp interacts with replication factors and the cell cycle protein, p21(Cip1/Waf1) and functions as a molecular switch for DNA elongation. To understand how DNA replication is regulated through PCNA, elucidation of the precise mechanisms of these protein interactions is necessary.
Results:
Loop-region mutants in which human PCNA sequences were substituted with the corresponding Saccharomyces cerevisiae PCNA regions were prepared. Analysis of their functions, along with previously prepared alanine scanning mutants, demonstrated that some loops interact with DNA polymerase delta (pol delta) and replication factor C (RFC). The p21 binding sites of PCNA, mapped by affinity measurement of the mutant forms, found to be located within a distinct structure of the PCNA monomer, overlap with RFC- and pol delta-interaction sites. Competition between p21 and pol delta or RFC for binding to PCNA results in efficient inhibition of its stimulation of pol delta DNA synthesis and RFC ATPase but not of PCNA loading on DNA by RFC.
Conclusions:
Semi-saturated amounts of p21 selectively block formation of the active pol delta complex but not the RFC-PCNA complex at 3'-ends of DNA primers. This differential effect may explain the specific inhibition of DNA replication by p21.
Insights
The cell cycle protein p21 selectively inhibits DNA replication by blocking the interaction between PCNA and DNA polymerase delta, but not PCNA loading by RFC.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Biology
Background:
- Proliferating Cell Nuclear Antigen (PCNA) is a crucial eukaryotic DNA sliding clamp regulating DNA elongation.
- PCNA interacts with replication factors and cell cycle proteins like p21(Cip1/Waf1), acting as a molecular switch.
- Understanding PCNA-mediated regulation of DNA replication requires detailed knowledge of its protein interactions.
Purpose of the Study:
- To elucidate the precise mechanisms of protein interactions involving PCNA in DNA replication regulation.
- To investigate how the cell cycle protein p21 inhibits DNA replication through its interaction with PCNA.
Main Methods:
- Construction and functional analysis of PCNA loop-region mutants and alanine scanning mutants.
- Affinity measurements to map p21 binding sites on PCNA.
- Assays to evaluate the effects of p21 on PCNA interactions with DNA polymerase delta (pol delta) and replication factor C (RFC).
Main Results:
- Specific PCNA loops were identified as interaction sites for pol delta and RFC.
- p21 binding sites on PCNA were found to overlap with pol delta and RFC interaction sites.
- Competition between p21 and pol delta/RFC for PCNA binding inhibited pol delta DNA synthesis and RFC ATPase activity, but not PCNA loading by RFC.
Conclusions:
- Semi-saturated p21 concentrations selectively inhibit the formation of the active pol delta-PCNA complex at DNA primer 3'-ends.
- This selective inhibition of complex formation, rather than PCNA loading, by p21 may explain its specific role in blocking DNA replication.