The Interdomain Connector Loop of Human PCNA Is Involved in a Direct Interaction with Human Polymerase δ

Proliferating cell nuclear antigen (PCNA) is required for processive DNA synthesis catalyzed by DNA polymerase δ (pol δ) and polymerase ε. We have shown that the epitope of a human PCNA inhibitory monoclonal antibody (74B1), which inhibits the PCNA stimulation of DNA synthesis catalyzed by pol δ, maps to residues 121–135, which overlap the interdomain connector loop of PCNA (residues 119–133). We have mutagenized residues 122–133 of human PCNA. The mutant proteins were expressed in Escherichia coli and purified to near-homogeneity. The interactions of the mutants with antibody 74B1 were examined; mutation of Gly-127 abolished the recognition by antibody 74B1 in a Western blot analysis, confirming the epitope assignment of 74B1. Mutations of Val-123, Leu-126, Gly-127, and Ile-128 affected the ability of PCNA to stimulate DNA synthesis by pol δ in several different assays. These mutations affected the interactions between PCNA and pol δ as determined by enzyme-linked immunosorbent assays. These mutants were also affected in their abilities to form a ternary complex with a DNA template-primer, as determined by electrophoretic mobility gel shift assays. The findings show that the interdomain connector loop region is involved in binding of pol δ. This same region is involved in the binding of p21, and our findings support the view that the mechanism of inhibition of DNA synthesis by p21 is due to a competition for PCNA binding to pol δ. Proliferating cell nuclear antigen (PCNA) is required for processive DNA synthesis catalyzed by DNA polymerase δ (pol δ) and polymerase ε. We have shown that the epitope of a human PCNA inhibitory monoclonal antibody (74B1), which inhibits the PCNA stimulation of DNA synthesis catalyzed by pol δ, maps to residues 121–135, which overlap the interdomain connector loop of PCNA (residues 119–133). We have mutagenized residues 122–133 of human PCNA. The mutant proteins were expressed in Escherichia coli and purified to near-homogeneity. The interactions of the mutants with antibody 74B1 were examined; mutation of Gly-127 abolished the recognition by antibody 74B1 in a Western blot analysis, confirming the epitope assignment of 74B1. Mutations of Val-123, Leu-126, Gly-127, and Ile-128 affected the ability of PCNA to stimulate DNA synthesis by pol δ in several different assays. These mutations affected the interactions between PCNA and pol δ as determined by enzyme-linked immunosorbent assays. These mutants were also affected in their abilities to form a ternary complex with a DNA template-primer, as determined by electrophoretic mobility gel shift assays. The findings show that the interdomain connector loop region is involved in binding of pol δ. This same region is involved in the binding of p21, and our findings support the view that the mechanism of inhibition of DNA synthesis by p21 is due to a competition for PCNA binding to pol δ. DNA polymerase δ (pol δ) 1The abbreviations used are: pol δ, polymerase δ; PCNA, proliferating cell nuclear antigen; RF-C, replication factor C; RPA, replication protein A; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; EMSA, electrophoretic mobility shift assay; Bis-Tris, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane. 1The abbreviations used are: pol δ, polymerase δ; PCNA, proliferating cell nuclear antigen; RF-C, replication factor C; RPA, replication protein A; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; EMSA, electrophoretic mobility shift assay; Bis-Tris, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane. is a central enzyme involved in the replication of mammalian chromosomal DNA and is also involved in DNA repair (1Zeng X.R. Jiang Y. Zhang S.J. Hao H. of in replication the of pol δ, as as of the complex of proteins that involved in DNA replication is that pol is involved in the pol δ is involved in synthesis and also for of synthesis of the of a DNA polymerase is the ability for processive synthesis of This ability is by which as These form that the DNA and also with the This in Escherichia coli is by the and in mammalian by PCNA cell nuclear which is factor for the DNA replication and as a factor for pol δ The for the of the of coli and PCNA by the of their which to proteins that the DNA PCNA is a of the a PCNA is DNA by the of by the of pol δ between PCNA and pol δ to form a ternary pol is the involved in the of PCNA with pol is also in DNA repair and cell Y. Zhang H. PCNA shown to with p21 H. S.J. The is a cell which inhibits protein and also shown to DNA synthesis in H. S.J. The mechanism for the shown to due to the ability of p21 to to human PCNA p21, the repair of pol δ S.J. that a the of p21 DNA replication and nuclear repair H. The of the of p21 with PCNA determined the by of the of a complex of with a the of p21 that inhibitory monoclonal antibody (74B1), inhibits the ability of to stimulate DNA synthesis catalyzed by pol δ in as a The epitope of antibody to residues of human PCNA Jiang Y. Zhang The that region is in the loop that the in the PCNA a of loop region to the for the interactions of with pol δ by the of in with electrophoretic mobility and have of the residues between and to the in PCNA that involved in with pol δ. The of region our assignment of the epitope of the inhibitory monoclonal antibody 74B1 to residues of by the of Jiang Y. Zhang that a for the of the their the assignment of the epitope of antibody 74B1. of the PCNA Western with the 74B1 antibody This of of the mutant with the monoclonal antibody 74B1 that the region between residues 122–133 of the epitope for 74B1. The that connector loop residues and Gly-127 to also involved in the between antibody 74B1 and is that mutation of the Gly-127 a the between and pol δ. mutation of of the of a to the of the epitope that is required for with the monoclonal our findings the epitope assignment of monoclonal antibody 74B1 and our in of the of the inhibitory of antibody as due to binding to a region involved in with pol δ. These findings the inhibition of PCNA stimulation of pol δ by antibody to due to with the binding of PCNA to pol of the of mutations residues 122–133 in the interdomain connector of PCNA the with pol δ in several These that region of is involved in binding to pol δ. of Gly-127 and Ile-128 in in the stimulation of pol δ. and in the stimulation of pol δ This of to due to a between pol δ and PCNA by in the of Gly-127 and that the ability of the PCNA and pol δ to form a ternary complex with a to a of the of mutant δ as complex with the same mutants also with and findings in This that mutations of Gly-127 and Ile-128 a the ability of human PCNA to stimulate the human pol δ for the of mutation of Gly-127 is that a in the of the interdomain connector loop of which region of to pol δ. The of the PCNA mutants with monoclonal antibody 74B1 is with of mutations in the interdomain connector loop that PCNA δ in a The of the ability of the PCNA mutants to stimulate in DNA synthesis catalyzed by pol δ due to the of the of inhibition of the of PCNA of the binding of pol δ to the in the of the δ complex the DNA that the is and in the of the mutants due to The PCNA mutants with to the of PCNA with the the that the of the PCNA mutants to stimulate pol δ due to a in the between and PCNA, is required in the assays. is also that the in the ability to stimulate DNA synthesis is the of in the of PCNA pol δ the DNA and that the of PCNA mutants were a of the of the binding of pol δ to of ternary pol the and The of the pol complex is determined by the between pol δ and PCNA, and the between the pol complex and the DNA of to the of the pol the PCNA mutants and that a in their ability to stimulate pol δ that the of PCNA mutants in the stimulation of pol δ the of with pol δ. mutants and which in the stimulation of pol δ of in is that the interactions between the pol complex and the DNA were that were by EMSA, to stimulate DNA synthesis of with a the of protein p21 (residues This that the p21 with the interdomain loop of PCNA that involved in the with the of between residues of p21 and residues of PCNA. region of is a residues Leu-126, and of the PCNA These with and form a that with residues of Gly-127, and form of a binding in the which the of and of p21 is a by residues and of the PCNA which with of the p21 the of p21 the mutants of PCNA that with pol δ is that is a between the residues mutation affected and involved in the is that the of p21 the inhibition of DNA pol δ is by a competition with PCNA H. S.J. The of the is as to a that the binding of pol δ to PCNA interactions as with p21 to PCNA, of region of with the interdomain loop of and with the The that a of pol δ is involved in the with PCNA is by that a to residues of pol δ is to PCNA of pol δ S.J. X.R. Zhang This is of a region in the between and DNA Zhang Hao H. have several of PCNA, have the of the interdomain connector loop in the with pol δ. the mutation of residues of to the that of residues have the the of PCNA H. of the mutants and in the stimulation of pol δ; residues in the the of the PCNA the residues that the of PCNA, pol δ were with and The for the stimulation of pol δ were for the and between and for the and The is of the residues in of the assays. of PCNA in which of residues were also the mutation a with pol δ. mutants in the loop and have PCNA, residues in the loop of PCNA that in and human PCNA. that mutation of and in abilities to stimulate pol δ, and that residues a in with pol δ. The between the and the of that is involved is of as mutation of of H. These show the of and PCNA stimulation of pol δ. is in the loop between and of which of the of is in the loop between and of which residues that form of the of of of the mutation of the of the which and a between residues and with pol δ is findings that the that involved in p21 binding also in the binding of pol δ. in and PCNA is in is a and human PCNA, is is to residues that form the a in p21 in and and human proteins to the same in PCNA and have the a of DNA polymerase δ in the also a mutation of in PCNA binding in and also to the interdomain connector loop region of PCNA. p21, the (residues is the binding with PCNA and as the and residues with the The in to our is the region of pol δ also a that as a of the is the region Zhang Hao H. the of (residues which the same of a with a for the findings that PCNA with a of proteins in the form of the of a for PCNA binding and the of the binding of p21 to PCNA the the of a of pol δ that to the of in the binding of the pol δ to PCNA, our that the of pol δ with PCNA. is is the that is for the of pol δ with PCNA. The enzyme used in purified by the and gel and the form that is Y. Zhang S.J. Zhang and Y. in our the pol δ in with PCNA in the of expressed coli Y. Jiang Y. Zhang Zhang S.J. Y. δ by of the and (pol δ in the by PCNA the stimulation by the the is that the of PCNA pol δ interactions the same PCNA is binding have for the of the region of PCNA that is involved in binding to pol δ to the interdomain connector loop of PCNA. This is and a of the epitope of inhibitory the of the which is also to the interdomain connector a for the that pol δ in a and a mechanism for the ability of p21 to DNA DNA polymerase δ (pol δ) 1The abbreviations used are: pol δ, polymerase δ; PCNA, proliferating cell nuclear antigen; RF-C, replication factor C; RPA, replication protein A; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; EMSA, electrophoretic mobility shift assay; Bis-Tris, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane. 1The abbreviations used are: pol δ, polymerase δ; PCNA, proliferating cell nuclear antigen; RF-C, replication factor C; RPA, replication protein A; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; EMSA, electrophoretic mobility shift assay; Bis-Tris, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane. is a central enzyme involved in the replication of mammalian chromosomal DNA and is also involved in DNA repair (1Zeng X.R. Jiang Y. Zhang S.J. Hao H. of in replication the of pol δ, as as of the complex of proteins that involved in DNA replication is that pol is involved in the pol δ is involved in synthesis and also for of synthesis of the of a DNA polymerase is the ability for processive synthesis of This ability is by which as These form that the DNA and also with the This in Escherichia coli is by the and in mammalian by PCNA cell nuclear which is factor for the DNA replication and as a factor for pol δ The for the of the of coli and PCNA by the of their which to proteins that the DNA PCNA is a of the a PCNA is DNA by the of by the of pol δ between PCNA and pol δ to form a ternary pol is the involved in the of PCNA with pol δ. PCNA is also in DNA repair and cell Y. Zhang H. PCNA shown to with p21 H. S.J. The is a cell which inhibits protein and also shown to DNA synthesis in H. S.J. The mechanism for the shown to due to the ability of p21 to to human PCNA p21, the repair of pol δ S.J. that a the of p21 DNA replication and nuclear repair H. The of the of p21 with PCNA determined the by of the of a complex of with a the of p21 that inhibitory monoclonal antibody (74B1), inhibits the ability of to stimulate DNA synthesis catalyzed by pol δ in as a The epitope of antibody to residues of human PCNA Jiang Y. Zhang The that region is in the loop that the in the PCNA a of loop region to the for the interactions of with pol δ by the of in with electrophoretic mobility and assays. have of the residues between and to the in PCNA that involved in with pol δ. The of region our assignment of the epitope of the inhibitory monoclonal antibody 74B1 to residues of by the of Jiang Y. Zhang that a for the of the their the assignment of the epitope of antibody 74B1. of the PCNA Western with the 74B1 antibody This of of the mutant with the monoclonal antibody 74B1 that the region between residues 122–133 of the epitope for 74B1. The that connector loop residues and Gly-127 to also involved in the between antibody 74B1 and is that mutation of the Gly-127 a the between and pol δ. mutation of of the of a to the of the epitope that is required for with the monoclonal our findings the epitope assignment of monoclonal antibody 74B1 and our in of the of the inhibitory of antibody as due to binding to a region involved in with pol δ. These findings the inhibition of PCNA stimulation of pol δ by antibody to due to with the binding of PCNA to pol of the of mutations residues 122–133 in the interdomain connector of PCNA the with pol δ in several These that region of is involved in binding to pol δ. of Gly-127 and Ile-128 in in the stimulation of pol δ. and in the stimulation of pol δ This of to due to a between pol δ and PCNA by in the of Gly-127 and that the ability of the PCNA and pol δ to form a ternary complex with a to a of the of mutant δ as complex with the same mutants also with and findings in This that mutations of Gly-127 and Ile-128 a the ability of human PCNA to stimulate the human pol δ for the of mutation of Gly-127 is that a in the of the interdomain connector loop of which region of to pol δ. The of the PCNA mutants with monoclonal antibody 74B1 is with of mutations in the interdomain connector loop that PCNA δ in a The of the ability of the PCNA mutants to stimulate in DNA synthesis catalyzed by pol δ due to the of the of inhibition of the of PCNA of the binding of pol δ to the in the of the δ complex the DNA that the is and in the of the mutants due to The PCNA mutants with to the of PCNA with the the that the of the PCNA mutants to stimulate pol δ due to a in the between and PCNA, is required in the assays. is also that the in the ability to stimulate DNA synthesis is the of in the of PCNA pol δ the DNA and that the of PCNA mutants were a of the of the binding of pol δ to of ternary pol the and The of the pol complex is determined by the between pol δ and PCNA, and the between the pol complex and the DNA of to the of the pol the PCNA mutants and that a in their ability to stimulate pol δ that the of PCNA mutants in the stimulation of pol δ the of with pol δ. mutants and which in the stimulation of pol δ of in is that the interactions between the pol complex and the DNA were that were by EMSA, to stimulate DNA synthesis of with a the of protein p21 (residues This that the p21 with the interdomain loop of PCNA that involved in the with the of between residues of p21 and residues of PCNA. region of is a residues Leu-126, and of the PCNA These with and form a that with residues of Gly-127, and form of a binding in the which the of and of p21 is a by residues and of the PCNA which with of the p21 the of p21 the mutants of PCNA that with pol δ is that is a between the residues mutation affected and involved in the is that the of p21 the inhibition of DNA pol δ is by a competition with PCNA H. S.J. The of the is as to a that the binding of pol δ to PCNA interactions as with p21 to PCNA, of region of with the interdomain loop of and with the The that a of pol δ is involved in the with PCNA is by that a to residues of pol δ is to PCNA of pol δ S.J. X.R. Zhang This is of a region in the between and DNA Zhang Hao H. have several of PCNA, have the of the interdomain connector loop in the with pol δ. the mutation of residues of to the that of residues have the the of PCNA H. of the mutants and in the stimulation of pol δ; residues in the the of the PCNA the residues that the of PCNA, pol δ were with and The for the stimulation of pol δ were for the and between and for the and The is of the residues in of the assays. of PCNA in which of residues were also the mutation a with pol δ. mutants in the loop and have PCNA, residues in the loop of PCNA that in and human PCNA. that mutation of and in abilities to stimulate pol δ, and that residues a in with pol δ. The between the and the of that is involved is of as mutation of of H. These show the of and PCNA stimulation of pol δ. is in the loop between and of which of the of is in the loop between and of which residues that form of the of of of the mutation of the of the which and a between residues and with pol δ is findings that the that involved in p21 binding also in the binding of pol δ. in and PCNA is in is a and human PCNA, is is to residues that form the a in p21 in and and human proteins to the same in PCNA and have the a of DNA polymerase δ in the also a mutation of in PCNA binding in and also to the interdomain connector loop region of PCNA. p21, the (residues is the binding with PCNA and as the and residues with the The in to our is the region of pol δ also a that as a of the is the region Zhang Hao H. the of (residues which the same of a with a for the findings that PCNA with a of proteins in the form of the of a for PCNA binding and the of the binding of p21 to PCNA the the of a of pol δ that to the of in the binding of the pol δ to PCNA, our that the of pol δ with PCNA. is is the that is for the of pol δ with PCNA. The enzyme used in purified by the and gel and the form that is Y. Zhang S.J. Zhang and Y. in our the pol δ in with PCNA in the of expressed coli Y. Jiang Y. Zhang Zhang S.J. Y. δ by of the and (pol δ in the by PCNA the stimulation by the the is that the of PCNA pol δ interactions the same PCNA is binding have for the of the region of PCNA that is involved in binding to pol δ to the interdomain connector loop of PCNA. This is and a of the epitope of inhibitory the of the which is also to the interdomain connector a for the that pol δ in a and a mechanism for the ability of p21 to DNA We have of the residues between and to the in PCNA that involved in with pol δ. The of region our assignment of the epitope of the inhibitory monoclonal antibody 74B1 to residues of by the of Jiang Y. Zhang that a for the of the their the assignment of the epitope of antibody 74B1. of the PCNA Western with the 74B1 antibody This of of the mutant with the monoclonal antibody 74B1 that the region between residues 122–133 of the epitope for 74B1. The that connector loop residues and Gly-127 to also involved in the between antibody 74B1 and is that mutation of the Gly-127 a the between and pol δ. mutation of of the of a to the of the epitope that is required for with the monoclonal our findings the epitope assignment of monoclonal antibody 74B1 and our in of the of the inhibitory of antibody as due to binding to a region involved in with pol δ. These findings the inhibition of PCNA stimulation of pol δ by antibody to due to with the binding of PCNA to pol δ. The of the of mutations residues 122–133 in the interdomain connector of PCNA the with pol δ in several These that region of is involved in binding to pol δ. of Gly-127 and Ile-128 in in the stimulation of pol δ. and in the stimulation of pol δ This of to due to a between pol δ and PCNA by in the of Gly-127 and that the ability of the PCNA and pol δ to form a ternary complex with a to a of the of mutant δ as complex with the same mutants also with and findings in This that mutations of Gly-127 and Ile-128 a the ability of human PCNA to stimulate the human pol δ for the of mutation of Gly-127 is that a in the of the interdomain connector loop of which region of to pol δ. The of the PCNA mutants with monoclonal antibody 74B1 is with The of the ability of the PCNA mutants to stimulate in DNA synthesis catalyzed by pol δ due to the of the of inhibition of the of PCNA of the binding of pol δ to the in the of the δ complex the DNA that the is and in the of the mutants due to The PCNA mutants with to the of PCNA with the the that the of the PCNA mutants to stimulate pol δ due to a in the between and PCNA, is required in the assays. is also that the in the ability to stimulate DNA synthesis is the of in the of PCNA pol δ the DNA and that the of PCNA mutants were a of the of the binding of pol δ to PCNA. The of ternary pol the and The of the pol complex is determined by the between pol δ and PCNA, and the between the pol complex and the DNA of to the of the pol the PCNA mutants and that a in their ability to stimulate pol δ that the of PCNA mutants in the stimulation of pol δ the of with pol δ. mutants and which in the stimulation of pol δ of in is that the interactions between the pol complex and the DNA were that were by EMSA, to stimulate DNA synthesis The of with a the of protein p21 (residues This that the p21 with the interdomain loop of PCNA that involved in the with the of between residues of p21 and residues of PCNA. region of is a residues Leu-126, and of the PCNA These with and form a that with residues of Gly-127, and form of a binding in the which the of and of p21 is a by residues and of the PCNA which with of the p21 the of p21 the mutants of PCNA that with pol δ is that is a between the residues mutation affected and involved in the is that the of p21 the inhibition of DNA pol δ is by a competition with PCNA H. S.J. The of the is as to a that the binding of pol δ to PCNA interactions as with p21 to PCNA, of region of with the interdomain loop of and with the The that a of pol δ is involved in the with PCNA is by that a to residues of pol δ is to PCNA of pol δ S.J. X.R. Zhang This is of a region in the between and DNA Zhang Hao H. have several of PCNA, have the of the interdomain connector loop in the with pol δ. the mutation of residues of to the that of residues have the the of PCNA H. of the mutants and in the stimulation of pol δ; residues in the the of the PCNA the residues that the of PCNA, pol δ were with and The for the stimulation of pol δ were for the and between and for the and The is of the residues in of the assays. of PCNA in which of residues were also the mutation a with pol δ. mutants in the loop and have PCNA, residues in the loop of PCNA that in and human PCNA. that mutation of and in abilities to stimulate pol δ, and that residues a in with pol δ. The between the and the of that is involved is of as mutation of of H. These show the of and PCNA stimulation of pol δ. is in the loop between and of which of the of is in the loop between and of which residues that form of the of of of the mutation of the of the which and a between residues and with pol δ is findings that the that involved in p21 binding also in the binding of pol δ. in and PCNA is in is a and human PCNA, is is to residues that form the a in p21 in and and human proteins to the same in PCNA and have the a of DNA polymerase δ in the also a mutation of in PCNA binding in and also to the interdomain connector loop region of PCNA. p21, the (residues is the binding with PCNA and as the and residues with the The in to our is the region of pol δ also a that as a of the is the region Zhang Hao H. the of (residues which the same of a with PCNA. a for the findings that PCNA with a of proteins in the form of the of a for PCNA binding and the of the binding of p21 to PCNA the the of a of pol δ that to the of in the binding of the pol δ to PCNA, our that the of pol δ with PCNA. is is the that is for the of pol δ with PCNA. The enzyme used in purified by the and gel and the form that is Y. Zhang S.J. Zhang and Y. in our the pol δ in with PCNA in the of expressed coli Y. Jiang Y. Zhang Zhang S.J. Y. δ by of the and (pol δ in the by PCNA the stimulation by the the is that the of PCNA pol δ interactions the same PCNA is binding have for the of the region of PCNA that is involved in binding to pol δ to the interdomain connector loop of PCNA. This is and a of the epitope of inhibitory the of the which is also to the interdomain connector a for the that pol δ in a and a mechanism for the ability of p21 to DNA