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Title: Proliferating cell nuclear antigen (PCNA) interacts with a meiosis-specific RecA homologues, Lim15/Dmc1, but does not stimulate its strand transfer activity

Abstract

PCNA is a multi-functional protein that is involved in various nuclear events. Here we show that PCNA participates in events occurring during early meiotic prophase. Analysis of protein-protein interactions using surface plasmon resonance indicates that Coprinus cinereus PCNA (CoPCNA) specifically interacts with a meiotic specific RecA-like factor, C. cinereus Lim15/Dmc1 (CoLim15) in vitro. The binding efficiency increases with addition of Mg{sup 2+} ions, while ATP inhibits the interaction. Co-immunoprecipitation experiments indicate that the CoLim15 protein interacts with the CoPCNA protein in vitro and in the cell extracts. Despite the interaction between these two factors, no enhancement of CoLim15-dependent strand transfer activity by CoPCNA was found in vitro. We propose that the interaction between Lim15/Dmc1 and PCNA mediates the recombination-associated DNA synthesis during meiosis.

Authors:
 [1];  [1];  [1];  [1];  [1];  [1];  [1];  [2];  [3]
  1. Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba-ken 278-8510 (Japan)
  2. Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba-ken 278-8510 (Japan). E-mail: kengo@rs.noda.tus.ac.jp
  3. Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N., A3-025, Seattle, WA 98109 (United States)
Publication Date:
OSTI Identifier:
20857970
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 352; Journal Issue: 4; Other Information: DOI: 10.1016/j.bbrc.2006.11.094; PII: S0006-291X(06)02511-3; Copyright (c) 2006 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; ANTIGENS; ATP; BIOSYNTHESIS; DNA; DNA REPLICATION; IN VITRO; MEIOSIS; MITOSIS; PROTEINS; RECOMBINATION

Citation Formats

Hamada, Fumika N., Koshiyama, Akiyo, Namekawa, Satoshi H., Ishii, Satomi, Iwabata, Kazuki, Sugawara, Hiroko, Nara, Takayuki Y., Sakaguchi, Kengo, and Sawado, Tomoyuki. Proliferating cell nuclear antigen (PCNA) interacts with a meiosis-specific RecA homologues, Lim15/Dmc1, but does not stimulate its strand transfer activity. United States: N. p., 2007. Web. doi:10.1016/j.bbrc.2006.11.094.
Hamada, Fumika N., Koshiyama, Akiyo, Namekawa, Satoshi H., Ishii, Satomi, Iwabata, Kazuki, Sugawara, Hiroko, Nara, Takayuki Y., Sakaguchi, Kengo, & Sawado, Tomoyuki. Proliferating cell nuclear antigen (PCNA) interacts with a meiosis-specific RecA homologues, Lim15/Dmc1, but does not stimulate its strand transfer activity. United States. doi:10.1016/j.bbrc.2006.11.094.
Hamada, Fumika N., Koshiyama, Akiyo, Namekawa, Satoshi H., Ishii, Satomi, Iwabata, Kazuki, Sugawara, Hiroko, Nara, Takayuki Y., Sakaguchi, Kengo, and Sawado, Tomoyuki. Fri . "Proliferating cell nuclear antigen (PCNA) interacts with a meiosis-specific RecA homologues, Lim15/Dmc1, but does not stimulate its strand transfer activity". United States. doi:10.1016/j.bbrc.2006.11.094.
@article{osti_20857970,
title = {Proliferating cell nuclear antigen (PCNA) interacts with a meiosis-specific RecA homologues, Lim15/Dmc1, but does not stimulate its strand transfer activity},
author = {Hamada, Fumika N. and Koshiyama, Akiyo and Namekawa, Satoshi H. and Ishii, Satomi and Iwabata, Kazuki and Sugawara, Hiroko and Nara, Takayuki Y. and Sakaguchi, Kengo and Sawado, Tomoyuki},
abstractNote = {PCNA is a multi-functional protein that is involved in various nuclear events. Here we show that PCNA participates in events occurring during early meiotic prophase. Analysis of protein-protein interactions using surface plasmon resonance indicates that Coprinus cinereus PCNA (CoPCNA) specifically interacts with a meiotic specific RecA-like factor, C. cinereus Lim15/Dmc1 (CoLim15) in vitro. The binding efficiency increases with addition of Mg{sup 2+} ions, while ATP inhibits the interaction. Co-immunoprecipitation experiments indicate that the CoLim15 protein interacts with the CoPCNA protein in vitro and in the cell extracts. Despite the interaction between these two factors, no enhancement of CoLim15-dependent strand transfer activity by CoPCNA was found in vitro. We propose that the interaction between Lim15/Dmc1 and PCNA mediates the recombination-associated DNA synthesis during meiosis.},
doi = {10.1016/j.bbrc.2006.11.094},
journal = {Biochemical and Biophysical Research Communications},
number = 4,
volume = 352,
place = {United States},
year = {Fri Jan 26 00:00:00 EST 2007},
month = {Fri Jan 26 00:00:00 EST 2007}
}
  • A stable stoichiometric complex of archaeal DNA polymerase with proliferating cell nuclear antigen (PCNA) was formed using a PCNA monomer mutant and the complex was successfully crystallized. Replicative DNA polymerase interacts with processivity factors, the β-subunit of DNA polymerase III or proliferating cell nuclear antigen (PCNA), in order to function with a long template DNA. The archaeal replicative DNA polymerase from Pyrococcus furiosus interacts with PCNA via its PCNA-interacting protein (PIP) motif at the C-terminus. The PCNA homotrimeric ring contains one PIP interacting site on each monomer and since the ring can accommodate up to three molecules simultaneously, formation ofmore » a stable stoichiometric complex of PCNA with its interacting protein has been difficult to control in vitro. A stable complex of the DNA polymerase with PCNA, using a PCNA monomer mutant, has been purified and crystallized. The best ordered crystal diffracted to 3.0 Å resolution using synchrotron radiation. The crystals belong to space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 225.3, b = 123.3, c = 91.3 Å.« less
  • Proliferating cell nuclear antigen (PCNA) protein is one of the central molecules responsible for decisions of life and death of the cell. The PCNA gene is induced by p53, while PCNA protein interacts with p53-controlled proteins Gadd45, MyD118, CR6 and, most importantly, p21, in the process of deciding cell fate. If PCNA protein is present in abundance in the cell in the absence of p53, DNA replication occurs. On the other hand, if PCNA protein levels are high in the cell in the presence of p53, DNA repair takes place. If PCNA is rendered non-functional or is absent or presentmore » in low quantities in the cell, apoptosis occurs. The evolution from prokaryotes to eukaryotes involved a change of function of PCNA from a 'simple' sliding clamp protein of the DNA polymerase complex to an executive molecule controlling critical cellular decision pathways. The evolution of multicellular organisms led to the development of multicellular processes such as differentiation, senescence and apoptosis. PCNA, already an essential molecule in the life of single cellular organisms, then became a protein critical for the survival of multicellular organisms.« less
  • The proliferating cell nuclear antigen (PCNA) gene codes for a protein that is necessary for cellular DNA synthesis and cell cycle progression. A functional promoter has been identified in the 5{prime} flanking region of the human PCNA gene. An abbreviated promoter was found to be equally efficient in directing transcription from a linked reporter, whether placed in the correct or reverse orientation in respect to the coding sequence. The reporter used was a cDNA of human thymidine kinase (TK), and the bidirectionality of the promoter was demonstrated by its ability to confer the TK{sup +} phenotype to TK{sup {minus}}ts13 cellsmore » and by the amount of specific message in RNA blots. The PvuII promoter placed between two coding sequences is capable of driving transcription simultaneously in both directions. Finally, in blots of RNA from human cells, two transcripts could be detected that hybridized to a sense riboprobe from the 5{prime} flanking region of the human PCNA gene. The authors conclude that the locus for the human PCNA gene contains a bidirectional promoter producing diverging transcripts.« less
  • Hoxc8 is a member of Hox family transcription factors that play crucial roles in spatiotemporal body patterning during embryogenesis. Hox proteins contain a conserved 61 amino acid homeodomain, which is responsible for recognition and binding of the proteins onto Hox-specific DNA binding motifs and regulates expression of their target genes. Previously, using proteome analysis, we identified Proliferating cell nuclear antigen (Pcna) as one of the putative target genes of Hoxc8. Here, we asked whether Hoxc8 regulates Pcna expression by directly binding to the regulatory sequence of Pcna. In mouse embryos at embryonic day 11.5, the expression pattern of Pcna wasmore » similar to that of Hoxc8 along the anteroposterior body axis. Moreover, Pcna transcript levels as well as cell proliferation rate were increased by overexpression of Hoxc8 in C3H10T1/2 mouse embryonic fibroblast cells. Characterization of 2.3 kb genomic sequence upstream of Pcna coding region revealed that the upstream sequence contains several Hox core binding sequences and one Hox-Pbx binding sequence. Direct binding of Hoxc8 proteins to the Pcna regulatory sequence was verified by chromatin immunoprecipitation assay. Taken together, our data suggest that Pcna is a direct downstream target of Hoxc8.« less
  • Highlights: Black-Right-Pointing-Pointer Proliferating Cell Nuclear Antigen (PCNA) is phosphorylated at Y114. Black-Right-Pointing-Pointer Phospho-Y114 of PCNA is not required for cell proliferation for normal growth. Black-Right-Pointing-Pointer MCE during adipogenesis is abolished in the lack of the phosphorylation. Black-Right-Pointing-Pointer Homozygous Y114F mice are resistant to high fat diet induced obesity. Black-Right-Pointing-Pointer Our results shed light on the interface between proliferation and differentiation. -- Abstract: Clonal proliferation is an obligatory component of adipogenesis. Although several cell cycle regulators are known to participate in the transition between pre-adipocyte proliferation and terminal adipocyte differentiation, how the core DNA synthesis machinery is coordinately regulated in adipogenesismore » remains elusive. PCNA (Proliferating Cell Nuclear Antigen) is an indispensable component for DNA synthesis during proliferation. Here we show that PCNA is subject to phosphorylation at the highly conserved tyrosine residue 114 (Y114). Replacing the Y114 residue with phenylalanine (Y114F), which is structurally similar to tyrosine but cannot be phosphorylated, does not affect normal animal development. However, when challenged with high fat diet, mice carrying homozygous Y114F alleles (PCNA{sup F/F}) are resistant to adipose tissue enlargement in comparison to wild-type (WT) mice. Mouse embryonic fibroblasts (MEFs) harboring WT or Y114F mutant PCNA proliferate at similar rates. However, when subjected to adipogenesis induction in culture, PCNA{sup F/F} MEFs are not able to re-enter the cell cycle and fail to form mature adipocytes, while WT MEFs undergo mitotic clonal expansion in response to the adipogenic stimulation, accompanied by enhanced Y114 phosphorylation of PCNA, and differentiate to mature adipocytes. Consistent with the function of Y114 phosphorylation in clonal proliferation in adipogenesis, fat tissues isolated from WT mice contain significantly more adipocytes than those isolated from PCNA{sup F/F} mice. This study identifies a critical role for PCNA in adipose tissue development, and for the first time identifies a role of the core DNA replication machinery at the interface between proliferation and differentiation.« less