Mirit I. Aladjem^1*, Stefania Pasa², Silvio Parodi², John N. Weinstein¹, Yves Pommier¹, and Kurt W. Kohn^1*

¹Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, USA.
²Laboratory of Experimental Oncology, National Institute of Cancer Research and University of Genoa, Genoa, Italy.

*Corresponding authors. E-mail: aladjemm{at}mail.nih.gov (M.I.A.) and kohnk{at}dc37a.nci.nih.gov (K.W.K).

• Description
  
  • Introduction. Background: Principles of Assembly of Replication Initiation Complexes
    
  • Map 1. Molecules Involved in DNA Replication (Complete Map)
    
  • Map 2. Molecular Interactions during Mitosis Prevent Licensing of DNA for Replication Prior to Cell Division (Mitosis)
    
  • Map 3. Modulation of DNA Replication by the ATM Pathway after Irradiation or Induction of Double-Stranded DNA Breaks (Radiation)
    
  • Map 4. Modulation of Molecular Interactions after Inhibition of Replication Elongation (Inhibitors)
    
• Map Key
  
  • Formal Definitions of the MIM Language
    
  • Map Symbols and Conventions
    
• Technical Details
  
  • Format
    
  • Requirements

Description
The interactive electronic MIMs (eMIMs) show the predominant molecular interactions involved in the regulation of cell division (Introduction). A complete map of all interactions is provided (Map 1). Additional maps represent the subset of interactions that occur during mitosis to prevent licensing of DNA replication prior to cell division (Map 2), the inhibition of the cell cycle in response to DNA damage caused by irradiation (Map 3), and inhibition of the cell cycle in response to drugs that inhibit replication elongation (Map 4). In addition, the definitions for interpreting the eMIMs are provided below and in the Map Symbols figure. Alternative sites for accession of the eMIMs include Interaction Replication Maps [(http://www.stefaniapasa.it/RepMap/), try this site if you are a Mac user and have difficulty with the maps available here] and Molecular Interactions Maps at the Genomics and Bioinformatics Group (http://discover.nci.nih.gov/mim). The maps available at these alternate sites have some differences from the maps available here including the links to other databases and descriptive information. The maps were created by Margot Sunshine, Hong Cao, and David Kane under the scientific direction of Mirit Aladjem, Stefania Pasa, Yves Pommier, John Weinstein, and Kurt Kohn.

Introduction. Background: Principles of Assembly of Replication Initiation Complexes. The initiation of DNA replication is a highly regulated process that integrates signals from the cell cycle machinery with chromatin components. The molecular interactions involved in DNA replication insure coordinated replication of the entire genome only once per cell cycle. The molecular pathways involved in replication can be separated into three clear components: licensing, initiation, and regulatory interactions, such as phosphorylation.

Licensing is a process involving the binding of several protein complexes to chromatin. By definition, licensing distinguishes between postmitotic chromosomes that are ready to be duplicated and post-S-phase chromosomes that have already passed through one round of DNA replication. In eucaryotes, a six-protein origin recognition complex (ORC) provides a platform for the binding of other licensing factors to chromatin. Data from yeast suggest that ORC binding sites include, but are not limited to, replication initiation sites. Some components of ORC bind to these sites throughout the cell cycle. Replication licensing involves binding of the ORC and DNA complex to another six- protein complex, the minichromosome maintenance (MCM) helicase. This binding only occurs in postmitotic cells and requires a coordinated pre-binding of a series of cell cycle regulatory factors, such as Cdc6 and Cdt1. Subcomplexes that form from either ORC or MCM contain targets for modification, primarily phosphorylation, by cell cycle regulators. The molecular interactions involving ORC, MCMs, and co-factors are described in the “licensing module”, which consists of coordinates E1 through G7 of Map 1.

Initiation of DNA replication requires binding of additional components to licensed chromatin. These include Cdc45, a protein subject to direct phosphorylation by two kinases that regulate cell cycle progression: cyclin dependent kinases (CDKs) and dbf4-dependent kinases (such as, Cdc7). Phosphorylation of Cdc45 by both kinases promotes its binding to the licensed chromatin/ORC/MCM complex, which leads to recruitment of other components of the replication machinery, and initiation of DNA replication.

Cell cycle-regulated modifications of the licensing and initiation proteins aims to insure replication at the right time during S phase and not at other times. For example, mitotic chromosomes do not initiate DNA replication, because all required licensing factors are not bound to the ORC. The lack of progression towards licensing is insured by several mechanisms (Map 2). First, mitotic kinases phosphorylate multiple sites on ORC and MCM complex subunits, preventing them from interacting with other subunits to form the complete licensing complex of ORC, MCM, and DNA. Second, the essential licensing co-factors, Cdc6 and Cdt1, are inactivated after completing their role in bringing together MCM and ORC complexes. These co-factors are targeted for degradation, cytoplasmic translocation, or both in yeast and vertebrates. The detailed mechanisms differ in various organisms, but in all cases, the absence of Cdt1 and Cdc6 from chromatin during the later stages of S phase prevents re-licensing of replicons that have already initiated DNA replication. The appearance of active Cdt1 and Cdc6 is delayed until after mitosis, insuring replication once per cell cycle.

Inhibition of DNA replication is the ultimate effect of converging regulatory “checkpoint” signals that serve to preserve genomic stability. The G1 and G2 checkpoints insure the inactivity of pathways leading to S phase, such as the activity of CDKs, through specific CDK inhibitors, such as p21 [for a detailed molecular interaction map of CDK inhibitors, see ( http://discover.nci.nih.gov/kohnk/kohnk.jsp)]. During S phase, DNA damage sensors inhibit initiation from unduplicated DNA sequences, while the system attempts to contain the damage suffered by sequences that had already been replicated. There are at least two parallel sensing and signaling pathways that accomplish this inhibition; each is activated by a different type of the DNA damage (Maps 3 and 4). Signals from both of these pathways converge at a late stage of the initiation cascade, preventing the binding of Ccd45 to licensed, unreplicated DNA. The mechanism(s) of this prevention are not yet clearly understood. The importance of checkpoint pathways is illustrated by the fact that proteins participating in these pathways are mutated in most human cancers. Elucidating the detailed interactions between molecules involved in cell cycle signaling and initiation of DNA replication is essential for understanding how cancer forms and progresses and may help design strategies to combat this disease.

Map 1. Molecules Involved in DNA Replication (Complete Map). DNA replication occurs only once per cell cycle due to a complex set of molecular interactions that can be divided into three pathways: Licensing, initiation, and regulatory interactions. During licensing, which occurs in late mitosis and early G1, postmitotic unreplicated chromatin binds several protein complexes. Initiation of DNA replication, which occurs throughout S phase, requires binding of additional factors that recruit the components of the replication machinery. After replication, the protein complexes that form the licensing factor dissociate from DNA, and the DNA is not a substrate for further initiations. Regulatory interactions occur on licensed or unlicensed chromatin in response to environmental conditions that determine whether cells proceed or halt their progression through the cell cycle. These interactions, which are mediated primarily through modifications of the phosphorylation status of proteins involved in licensing and initiation, determine whether replication will occur on a specific DNA template during a specific time of the cell cycle.

[Access Map 1 (Complete Map)]

Map 2. Molecular Interactions during Mitosis Prevent Licensing of DNA for Replication Prior to Cell Division (Mitosis). The complex formed by Cdk1 and the mitotic cyclin, cyclin B, phosphorylates proteins of the MCM complex, specifically MCM4, and possibly proteins of the ORC complex, specifically ORC1. These phosphorylation events inhibit the ability of MCM and ORC complexes to interact with other members of the licensing complex. In addition, during mitosis geminin binds and targets Cdt1 for degradation. The other accessory protein involved in licensing, Cdc6, leaves the nucleus in late G1 and does not reappear in the nucleus until after cell division. Because the accessory factors are not present, licensing cannot take place and DNA replication is blocked.

[Access Map 2 (Mitosis)]

Map 3. Modulation of DNA Replication by the ATM Pathway after Irradiation or Induction of Double-Stranded DNA Breaks (Irradiation). Exposure of cells to irradiation or to chemicals that cause double-stranded DNA breaks lead to the activation of the ATM pathway. The kinase ATM phosphorylates CHK2. The phosphorylated CHK2 catalyzes an inhibitory phosphorylation of Cdc25, a phosphatase required to dephosphorylate inhibitory sites on CDKs. Because CDKs cannot be activated, initiation of DNA replication and progression through the cell cycle cannot occur. In parallel, CHK2 also phosphorylates Dbf4-dependent kinases (DDKs, which in mammalian cells include the proteins hsk1 and dpf4) and this phosphorylation inhibits binding of the MCM complex to ORC-bound chromatin. Finally, there is some evidence suggesting a third pathway mediated by ATM that inhibits Cdc45 binding to licensed chromatin. All three sets of molecular interactions result in the inhibition of DNA replication from chromatin even if licensing (as measured by binding of the MCM complex) had already taken place. ATM kinase also activates the p53-mediated pathway that may lead to further kinase inhibition and apoptosis.

[Access Map 3 (Irradiation)]

Map 4. Modulation of Molecular Interactions after Inhibition of Replication Elongation (Inhibitors). During DNA synthesis, licensed replication origins require the activity of two sets of kinases, DDKs (hsk1) and CDKs (Cdk1 or Cdk2). Processes or agents that inhibit DNA replication activate the kinase ATR, which phosphorylates and activates CHK1. Activation of the kinase CHK1 leads to an inhibitory phosphorylation of Cdc25A and a stimulating phosphorylation of Wee1. Wee1 is a kinase that facilitates an inhibitory phosphorylation on CDKs; Cdc25A phosphorylation inhibits Cdc25A and does not allow the removal of the inhibitory phosphorylation on CDKs. Thus, phosphorylation of Cdc25A and of Wee1 results in CDKs remaining in a phosphorylated, inactive stage. In this context, CDKs cannot mediate initiation of DNA replication on licensed DNA.

[Access Map 4 (Inhibitors)]

Map Key
Formal Definitions of the MIM Language

1. A molecular species is either an elementary molecular species or a multimolecular complex.
2. An elementary molecular species is either a molecular species or group of species that is named within a round-cornered box (or “cartouche”). “Molecular species” refers to a molecule, such as a protein, having specific regulatory function, and named only in one place on a map. Small or ubiquitous molecules are marked without a surrounding box and may occur multiple times on a map.
3. A protein species may contain indicated sites and domains. Each site or domain can have its own interactions. The domains of a protein may be named within the protein’s cartouche in the order N- to C-terminus from left to right, in which case the protein itself is named immediately to the left of its cartouche. When the site or domain of an interaction is unknown, an interaction line may point to the protein name outside of the cartouche. Although not shown in the maps presented here, an example of a multidomain molecule is shown in the p53 map, which can be accessed from http://discover.nci.nih.gov/kohnk/kohnk.jsp.
4. A multimolecular complex is represented by a small filled circle (or “node”) on a binding interaction line.
5. An interaction generally consists of a reaction, consequences, and contingencies, all of which are encoded by the colors and shapes of the lines.
6. Reactions occur between molecular species. The reaction types and their symbols are listed in the Map Symbols figure. Reactions can include enzymatic processes, such as stoichometric conversion, covalent modification, binding interaction, or cleavage reaction.
7. Consequences are indicated as nodes on the lines that represent reactions or contingencies, that emerge from a node, representing a molecular species or an interaction. For example, a node on a binding-interaction line represents the consequence of the binding, which is the resulting molecular complex. A node on a protein modification line represents the consequence of the modification, which is the modified protein.
8. Contingencies are defined as stimulation or inhibition of a reaction or process (such as transcription), a requirement for species, or enzyme-dependence. Contingencies are shown associated with reactions or other contingencies. When multiple contingencies operate on a given reaction or contingency, logical operator symbols for “and” or “or” may be applied. An example for an “or” contingency can be seen in the cyclin box (Map 1, coordinates A5 through B6), in which Cdk1 can bind either to cyclin A or cyclin B, (but not to both cyclins at the same time) and Cdk2 can bind either to cyclin A or cyclin E.
9. When a molecular species has multiple interactions, it is assumed (unless otherwise specified in the annotation) that the interactions can be concurrent or occur in cis (that is, they involve the same molecule, rather than different molecules of the same species).
10. Interaction in trans, which is one molecule acting on another of the same species, is indicated by an interrupted interaction line with nodes at the ends of the line at the interruption point.

Map Symbols and Conventions
Right-click on the names of each of the molecular species represented in the map to reach sources of additional information (Entrez Nucleotide, Entrez Genome, PubMed, UniGene, GeneCards, Matchminer-aliases, Matchminer-location). To reach a brief description for a particular molecular interaction, click on the annotation number within the maps (for example, A45 in Map 1). Clicking on the numbers within the text of the annotation will lead to the cited references (for example, R89 in the A45 annotation of Map 1).

[Map Symbols Figure]

Technical Details
Format: Maps are rendered as scalable vector graphics (.svg)

Requirements: SVG Plug-in is available from Adobe (http://www.adobe.com/svg/)
The plug-in has been successfully tested with Internet Explorer (IE) 5.0, IE 5.5, IE 6, and Netscape 4.7 on Windows; and Netscape 4.7 on Mac OS8.6-9.2, or 10.1. The plug-in does not work with IE on a Mac or Netscape 6.0 on Windows.

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Citation: M. I. Aladjem, S. Pasa, S. Parodi, J. N. Weinstein, Y. Pommier, K. W. Kohn, Molecular interaction maps--A diagrammatic graphical language for bioregulatory networks. Sci. STKE 2004, pe8 (2004).

© 2004 American Association for the Advancement of Science