Initiation Of DNA Replication In Mammalian Chromosomes

Child Health And Human Development

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Abstract

Several years ago, we discovered that the behavior of ORC in mammalian cells differs significantly from its behavior in single cell eukaryotes such as yeast. Yeast ORCs consist of a stable complex of six different subunits that remain bound to chromatin throughout cell division and target specific DNA sequences. In contrast, mammalian ORCs consists of a stable core complex ORC(2-5) of Orc2 through Orc5 that interacts weakly with Orc1 and Orc6. Nevertheless, the association of Orc1 with ORC(2-5) is essential for prereplication complex assembly and DNA replication. In vitro, however, metazoan ORCs exhibit little affinity for specific DNA sequences other than a preference for asymmetric A:T-rich regions. Nevertheless, in the differentiated cells of mammals and flies, ORCs is localized at specific genomic sites that are coincident with DNA replication origins. Thus, the ability of ORC to activate a particular replication origin appears to depend on its ability to interact with DNA as it exists within the nucleus, an interaction that appears to be regulated by Orc1. [unreadable] [unreadable] During the past year, we established the basic features of the ORC cycle, a regulatory pathway we proposed previously that restricts initiation of DNA replication events so that genomes are duplicated once and only once each time a cell divides. The largest subunit (Orc1) regulates association of the stable ORC(2-5) core complex with replication origins in vivo, and that it does so through its BAH domain. This domain interacts with another, as yet unidentified, protein that is required for ORC binding to chromosomes as cells exit mitosis and begin a new cell division cycle. Moreover, if Orc1 is not bound to ORC(2-5), then it can induce apoptosis (programmed cell death). However, if the unbound Orc1 is either mono-ubiquitinated or phosphorylated (two normal cell cycle dependent modifications of Orc1), then the modified Orc1 is localized in the cytoplasm where it cannot initiate replication. This accounts for the fact that Orc1 is modified and in some cells degraded as the cells enter S-phase (period of DNA replication). In addition, the modified forms of Orc1 do not induce apoptosis. Thus, cell cycle modifications regulate Orc1 activity, and Orc1 activity regulates ORC activity, which regulates initiation of DNA replication.[unreadable] [unreadable] Further information is found at our web site (http://depamphilislab.nichd.nih.gov/).[unreadable] [unreadable] Specifics[unreadable] [unreadable] 1) Selection of initiation sites for DNA replication in eukaryotes is determined by the interaction between the origin recognition complex (ORC) and genomic DNA. In mammalian cells, this interaction appears to be regulated by Orc1, the only ORC subunit that contains a BAH domain. Since BAH domains mediate protein-protein interactions, the human Orc1 BAH domain was mutated, and the mutant proteins expressed in human cells to determine their affects on ORC function. The BAH domain was not required for nuclear localization of Orc1, association of Orc1 with other ORC subunits, or selective degradation of Orc1 during S-phase. It did, however, facilitate reassociation of Orc1 with chromosomes during the M to G1-phase transition, and it was required for binding Orc1 to the Epstein-Barr virus oriP and stimulating oriP dependent plasmid DNA replication. Moreover, the BAH domain affected Orc1s ability to promote binding of Orc2 to chromatin as cells exit mitosis. Thus, the BAH domain in human Orc1 facilitates its ability to activate replication origins in vivo by promoting association of ORC with chromatin.[unreadable] [unreadable] 2) Previous studies have suggested that the activity of the mammalian origin recognition complex (ORC) is regulated by cell cycle dependent changes in its Orc1 subunit. Here we show that Orc1 modifications such as monoubiquitination and hyperphosphorylation that occur normally during S and G2/M phases, respectively, can cause Orc1 to accumulate in the cytoplasm. This would suppress reassembly of prereplication complexes until mitosis is complete. In the absence of these modifications, transient expression of Orc1 rapidly induced p53-independent apoptosis, and Orc1 accumulated perinuclearly rather than uniformly throughout the nucleus. This behavior mimicked the increased concentration and perinuclear accumulation of endogenous Orc1 in apoptotic cells that arise spontaneously in proliferating cell cultures. Remarkably, expression of Orc1 in the presence of an equivalent amount of Orc2, the only ORC subunit that did not induce apoptosis, prevented induction of apoptosis and restored uniform nuclear localization of Orc1. This would promote assembly of ORC:chromatin sites, such as occurs during the M to G1-phase transition. These results provide direct evidence in support of the regulatory role proposed for Orc1, and suggest that aberrant DNA replication during mammalian development could result in apoptosis through the appearance of unmodified Orc1.[unreadable] [unreadable] 3) Eukaryotic DNA replication begins with the binding of a six subunit origin recognition complex (ORC) to DNA. To study the assembly and function of mammalian ORC proteins in their native environment, HeLa cells were constructed that constitutively expressed an epitope tagged, recombinant human Orc2 subunit that had been genetically altered. Analysis of these cell lines revealed that Orc2 contains a single ORC assembly domain that is required in vivo for interaction with all other ORC subunits, as well as two nuclear localization signals (NLSs) that are required for ORC accumulation in the nucleus. The recombinant Orc2 existed in the nucleus either as an ORC2-5 or ORC1-5 complex; no other combinations of ORC subunits were detected. Moreover, only ORC1-5 was bound to the chromatin fraction, suggesting that Orc1 is required in vivo to load ORC2-5 onto chromatin. Surprisingly, recombinant Orc2 suppressed expression of endogenous Orc2, revealing that mammalian cells limit the intracellular level of Orc2, and thereby limit the amount of ORC2-5 in the nucleus. Since this suppression required only the ORC assembly and NLS domains, these domains appear to constitute the functional domain of Orc2. [unreadable] [unreadable] 4) I was privleged to act as editor for a book in which 72 leaders in the field of DNA replication described the various aspects of cellular and viral DNA replication and their relationships to human disease. This book marks a the first such effort to link the fundamental process of genome duplication to genetically inherited diseases in humans.