Abstract
The DNA polymerase accessory factor proliferating cell nuclear antigen (PCNA) has been caught in interaction with an ever increasing number of proteins. To characterize the sites and functions of some of these interactions, we constructed four mutants of human PCNA and analysed them in a variety of assays. By targeting loops on the surface of the PCNA trimer and changing three or four residues at a time to alanine, we found that a region including part of the domain-connecting loop of PCNA and loops on one face of the trimer, close to the C-termini, is involved in binding to all of the following proteins: DNA polymerase delta, replication factor C, the flap endonuclease Fen1, the cyclin dependent kinase inhibitor p21 and DNA ligase I. An inhibition of DNA ligation caused by the interaction of PCNA with DNA ligase I was found, and we show that DNA ligase I and Fen1 can inhibit DNA synthesis by DNA polymerase delta/PCNA. We demonstrate that PCNA must be located below a 5' flap on a forked template to stimulate Fen1 activity, and considering the interacting region on PCNA for Fen1, this suggests an orientation for PCNA during DNA replication with the C-termini facing forwards, in the direction of DNA synthesis.
Publication types
-
Research Support, Non-U.S. Gov't
MeSH terms
-
Amino Acid Sequence
-
Animals
-
Cyclin-Dependent Kinase Inhibitor p21
-
Cyclins / metabolism
-
DNA Ligase ATP
-
DNA Ligases / metabolism
-
DNA Polymerase III / metabolism
-
DNA Replication*
-
DNA-Binding Proteins / metabolism
-
Endodeoxyribonucleases / metabolism
-
Enzyme Inhibitors / metabolism
-
Exodeoxyribonuclease V
-
Exodeoxyribonucleases / metabolism
-
Flap Endonucleases*
-
Homeodomain Proteins*
-
Humans
-
Minor Histocompatibility Antigens
-
Models, Molecular
-
Molecular Sequence Data
-
Mutagenesis
-
Proliferating Cell Nuclear Antigen / chemistry
-
Proliferating Cell Nuclear Antigen / genetics
-
Proliferating Cell Nuclear Antigen / metabolism*
-
Protein Folding
-
Proto-Oncogene Proteins c-bcl-2*
-
Replication Protein C
-
Repressor Proteins*
-
Saccharomyces cerevisiae Proteins*
-
Structure-Activity Relationship
Substances
-
BCL2-related protein A1
-
CDKN1A protein, human
-
Cyclin-Dependent Kinase Inhibitor p21
-
Cyclins
-
DNA-Binding Proteins
-
Enzyme Inhibitors
-
Homeodomain Proteins
-
LIG1 protein, human
-
MATA1 protein, S cerevisiae
-
Minor Histocompatibility Antigens
-
Proliferating Cell Nuclear Antigen
-
Proto-Oncogene Proteins c-bcl-2
-
Repressor Proteins
-
Saccharomyces cerevisiae Proteins
-
DNA Polymerase III
-
Endodeoxyribonucleases
-
Exodeoxyribonucleases
-
Flap Endonucleases
-
FEN1 protein, human
-
Exodeoxyribonuclease V
-
Replication Protein C
-
DNA Ligases
-
DNA Ligase ATP