Functional sites of human PCNA which interact with p21 (Cip1/Waf1), DNA polymerase delta and replication factor C.
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.
Oku
T
T
Faculty of Biological Science, Nara Institute of Science and Technology, Takayama, Ikoma, Japan.
Ikeda
S
S
Sasaki
H
H
Fukuda
K
K
Morioka
H
H
Ohtsuka
E
E
Yoshikawa
H
H
Tsurimoto
T
T
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D001665:Binding Sites / Q000502:physiology; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000378:metabolism*; D004247:DNA / Q000096:biosynthesis; D004258:DNA Polymerase III / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D018398:Homeodomain Proteins; D006801:Humans; D015778:Minor Histocompatibility Antigens; D009154:Mutation; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D051818:Replication Protein C; D012097:Repressor Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins
C096816:BCL2-related protein A1; C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C096063:MATA1 protein, S cerevisiae; D015778:Minor Histocompatibility Antigens; D018809:Proliferating Cell Nuclear Antigen; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D004258:DNA Polymerase III; D000251:Adenosine Triphosphatases; D051818:Replication Protein C