ABSTRACT
The origin recognition complex (ORC) in yeast is a complex of six tightly associated subunits essential for the initiation of DNA replication. Human ORC subunits are nuclear in proliferating cells and in proliferative tissues like the testis, consistent with a role of human ORC in DNA replication. Orc2, Orc3, and Orc5 also are detected in non-proliferating cells like cardiac myocytes, adrenal cortical cells, and neurons, suggesting an additional role of these proteins in non-proliferating cells. Although Orc2-5 co-immunoprecipitate with each other under mild extraction conditions, a holo complex of the subunits is difficult to detect. When extracted under more stringent extraction conditions, several of the subunits co-immunoprecipitate with stoichiometric amounts of other unidentified proteins but not with any of the known ORC subunits. The variation in abundance of individual ORC subunits (relative to each other) in several tissues, expression of some subunits in non-proliferating tissues, and the absence of a stoichiometric complex of all the subunits in cell extracts indicate that subunits of human ORC in somatic cells might have activities independent of their role as a six subunit complex involved in replication initiation. Finally, all ORC subunits remain consistently nuclear, and Orc2 is consistently phosphorylated through all stages of the cell cycle, whereas Orc1 is selectively phosphorylated in mitosis.
Subject(s)
DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Blotting, Northern , Blotting, Western , Cell Cycle , Cell Division , Cell Nucleus/metabolism , Chromatography, Gel , DNA Replication , HeLa Cells , Humans , Immunohistochemistry , Male , Microscopy, Fluorescence , Mitosis , Origin Recognition Complex , Phosphorylation , Precipitin Tests , Testis/metabolism , Tissue Distribution , Tumor Cells, CulturedABSTRACT
Regulated initiation of DNA replication relies on the firing of initiator proteins that bind specifically to origin DNA. The discovery of the first eukaryotic initiator, the Saccharomyces cerevisiae Origin Recognition Complex (ORC) has allowed us to discern some aspects of how the onset of replication is regulated. However, understanding the specifics of replication in metazoan organisms can only be achieved by directly addressing these questions in animal cells. This review deals with the current state of knowledge on the metazoan Origin Recognition Complex, its composition and regulation in higher eukaryotes, its role in the initiation of replication and beyond replication, and its possible connection with human pathology.
Subject(s)
DNA Replication , DNA-Binding Proteins/physiology , Eukaryotic Cells/physiology , Saccharomyces cerevisiae Proteins , Animals , Cell Cycle Proteins/metabolism , Chromatin/metabolism , DNA-Binding Proteins/metabolism , Gene Expression Regulation , Humans , Origin Recognition Complex , Replication Origin , Saccharomyces cerevisiae/geneticsABSTRACT
The Drosophila latheo (lat) gene was identified in a behavioral screen for olfactory memory mutants. The original hypomorphic latP1 mutant (Boynton and Tully, 1992) shows a structural defect in adult brain. Homozygous lethal lat mutants lack imaginal discs, show little cell proliferation in the CNS of third instar larvae, and die as early pupae. latP1 was cloned, and all of the above mentioned defects of hypomorphic or homozygous lethal lat mutants were rescued with a lat+ transgene. lat encodes a novel protein with homology to a subunit of the origin recognition complex (ORC). Human and Drosophila LAT both associate with ORC2 and are related to yeast ORC3, suggesting that LAT functions in DNA replication during cell proliferation.
Subject(s)
DNA-Binding Proteins/genetics , Drosophila Proteins , Drosophila/genetics , Memory/physiology , Mutation/genetics , Neurons/pathology , Olfactory Pathways/physiopathology , Amino Acid Sequence/genetics , Animals , Animals, Genetically Modified , Brain/abnormalities , Brain/pathology , Brain/physiopathology , Cell Division/physiology , Central Nervous System/pathology , Congenital Abnormalities/genetics , Drosophila/growth & development , Memory Disorders/genetics , Molecular Sequence Data , Mutation/physiology , Origin Recognition Complex , Pupa/physiology , Sequence Homology, Amino Acid , Transcription, Genetic/genetics , Transgenes/physiologyABSTRACT
A new member of the human origin recognition complex (ORC) was cloned and identified as ORC5L. HsORC5p is a 50-kDa protein whose sequence is 38% identical and 62% similar to ORC5p from Drosophila melanogaster. Two alleles of ORC5L were identified, one with and one without an evolutionarily conserved purine nucleotide binding motif. HsORC5p is precipitated from cell extracts with HsORC2p and HsORC4p, indicating that it is part of the putative human ORC. The bulk of HsORC5p is in an insoluble nuclear fraction, whereas the other known human ORC subunits (HsORC1p, HsORC2p, and HsORC4p) are easily extracted in the nuclear-soluble fractions and in S100 (HsORC1p). In addition, we identified an alternatively spliced mRNA from the same locus (HsORC5T). HsORC5Tp also formed a complex with HsORC4p but not with HsORC2p, suggesting it may play a regulatory role in the assembly of different ORC subcomplexes. HsORC5, HsORC5T, and HsORC4 transcripts are abundant in spleen, ovary, and prostate in addition to tissues with high levels of DNA replication like testes and colon mucosa, implicating the human ORC proteins in functions besides DNA replication. Finally, the gene for ORC5L is located at chromosome 7, band q22, in the minimal region deleted in 10% of uterine leiomyomas and in 10-20% of acute myeloid leukemias and myelodysplastic syndromes.
Subject(s)
DNA-Binding Proteins/genetics , Gene Deletion , Leiomyoma/genetics , Leukemia, Myeloid/genetics , Myelodysplastic Syndromes/genetics , Uterine Neoplasms/genetics , Alternative Splicing , Amino Acid Sequence , Base Sequence , Cell Cycle , Chromosomes, Human, Pair 7 , Cloning, Molecular , DNA Replication , Female , Humans , Male , Molecular Sequence Data , Origin Recognition Complex , Replication Origin , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Subcellular Fractions/chemistry , Tissue DistributionABSTRACT
A new member of human origin recognition complex (ORC) has been cloned and identified as the human homologue of Saccharomyces cerevisiae ORC4. HsORC4 is a 45-kDa protein encoded by a 2.2-kilobase mRNA whose amino acid sequence is 29% identical to ScORC4. HsORC4 has a putative nucleotide triphosphate binding motif that is not seen in ScORC4. HsORC4P also reveals an unsuspected homology to the ORC1-Cdc18 family of proteins. HsORC4 mRNA expression and protein levels remain constant through the cell cycle. HsORC4P is coimmunoprecipitated from cell extracts with another subunit of human ORC, HsORC2P, consistent with it being a part of the putative human origin recognition complex.