DNA replication, development and cancer: a homeotic connection?

The homeotic proteins are transcription factors, highly conserved in metazoan organisms, exerting a pivotal role in development and differentiation. They individually display a loose specificity for the DNA sequence they can bind, but operate mainly in multi-molecular associations that assure their target and function specificity. Homeotic proteins are known to play a role in the positive or negative regulation of cell proliferation. Furthermore, many homeotic proteins are actually proto-oncogenes, since different translocations involving their genes cause tumors, particularly in the hematopoietic system. A one-hybrid screen to detect proteins with affinity for the lamin B2 replication origin identified three homeotic proteins, namely HoxA13, HoxC10 and HoxC13. Recent data demonstrate that the HoxC13 oncoprotein specifically associates with replication foci and binds in vitro and in vivo to several human DNA replication origins. Moreover, Hox proteins interact with geminin, a regulator of cell cycle progression, and control the interaction of this protein with the DNA replication licensing factor Ctd1. Thus, the homeotic proteins, by participating directly in the function of DNA replication origins, may provide a direct link between the accurate regulation of DNA replication required by the morphogenetic program and the deregulation of this process typical of cancer.

Homeotic proteins participate in the function of human-DNA replication origins.

Recent evidence points to homeotic proteins as actors in the crosstalk between development and DNA replication. The present work demonstrates that HOXC13, previously identified as a new member of human DNA replicative complexes, is a stable component of early replicating chromatin in living cells: it displays a slow nuclear dynamics due to its anchoring to the DNA minor groove via the arginine-5 residue of the homeodomain. HOXC13 binds in vivo to the lamin B2 origin in a cell-cycle-dependent manner consistent with origin function; the interaction maps with nucleotide precision within the replicative complex. HOXC13 displays in vitro affinity for other replicative complex proteins; it interacts also in vivo with the same proteins in a cell-cycle-dependent fashion. Chromatin-structure modifying treatments, disturbing origin function, reduce also HOXC13-origin interaction. The described interactions are not restricted to a single origin nor to a single homeotic protein (also HOXC10 binds the lamin B2 origin in vivo). Thus, HOX complexes probably contribute in a general, structure-dependent manner, to origin identification and assembly of replicative complexes thereon, in presence of specific chromatin configurations.

The homeotic protein HOXC13 is a member of human DNA replication complexes.

The homeotic (and oncogenic) HOXC13 protein was shown to have an affinity for a DNA fragment corresponding to the sequence covered by the pre-replicative complex of the human lamin B2 replication origin. We show here that HOXC13 is a member of human replicative complexes. Our fluorescent fusion-protein data demonstrate that it co-localizes with replication foci of early-S cells and that this peculiar behaviour is driven by the homeodomain. By ChIP analysis we also show that HOXC13 binds the lamin B2 replication origin and the origins located near the TOP1 and MCM4 genes in asynchronously growing cells, whereas it does not bind these origins in G(0) resting cells, consistently with its involvement in origin function.

Molecular mechanics and DNA replication regulation.

Magnetic and optical tweezers are providing novel insights on the structure, energetics, and functional dynamics of biological macromolecules. The modulation of DNA topology has provided very appropriate opportunities to study with these technologies the energetic and mechanistic features of the action of DNA topoisomerases, the enzymes that maintain the physiological level of negative superhelicity in the genome. Modulation of the superhelical state of the DNA replication origins is essential for the initiation of DNA synthesis in prokaryotes and eukaryotes. The results obtained recently from independent studies of two different groups combine to give new insights on the topological aspects of this process. With magnetic tweezers it was shown that the inhibition of human topoisomerase I by camptothecin freezes the drug-enzyme-DNA complex and specifically forbids the relaxation of positive supercoils; a study of the in vivo role of topoisomerase I on the activation of a human origin showed that this process is forbidden when the enzyme is frozen on the origin DNA by camptothecin. The inhibition of the relaxation of positive supercoils, probably introduced by the proteins performing origin activation, is therefore lethal for this process. Thus, the use of advanced physical technologies provides insights on an essential biological process.

Molecular and structural transactions at human DNA replication origins.

The DNA replication origins of metazoan genomes are the sites of complex sequence-specific protein-DNA interactions determining their precise cycle of activation and deactivation, once only along each cell cycle. Some of the involved proteins have been identified (and particularly the essential six-protein Origin Recognition Complex, ORC) thanks to their homology with the proteins identified in yeast. Whereas in the latter organism ORC has a specific affinity for an origin consensus, metazoan (and human) ORC shows no sequence specificity and no origin consensus is identifiable in their genomes. The modulation of topology around the origin sequence plays an essential role in the function of the human lamin B2 origin and the two topoisomerases interact specifically with it in a cell-cycle modulated way. The two enzymes are never present on the origin at the same time and compete, in different moments of the cell cycle, with the ORC2 subunit for the same sites in the origin area. The topoisomerases could give essential contributions to origin definition, as demonstrated by their capacity to bind specifically, in vitro the lamin B2 origin, either alone (topoisomerase I) or in a multi-protein complex (topoisomerase II). They also play critical roles in the origin activation-deactivation cycle, topoisomerase II probably contributing to attain and/or maintain a topological status fit for prereplicative complex assembly and topoisomerase I allowing the topological adaptations necessary for initiation of bi-directional synthesis.

Functional interactions of DNA topoisomerases with a human replication origin.

The human DNA replication origin, located in the lamin B2 gene, interacts with the DNA topoisomerases I and II in a cell cycle-modulated manner. The topoisomerases interact in vivo and in vitro with precise bonds ahead of the start sites of bidirectional replication, within the pre-replicative complex region; topoisomerase I is bound in M, early G1 and G1/S border and topoisomerase II in M and the middle of G1. The Orc2 protein competes for the same sites of the origin bound by either topoisomerase in different moments of the cell cycle; furthermore, it interacts on the DNA with topoisomerase II during the assembly of the pre-replicative complex and with DNA-bound topoisomerase I at the G1/S border. Inhibition of topoisomerase I activity abolishes origin firing. Thus, the two topoisomerases are closely associated with the replicative complexes, and DNA topology plays an essential functional role in origin activation.

Localization of proteins bound to a replication origin of human DNA along the cell cycle.

The proteins bound in vivo at the human lamin B2 DNA replication origin and their precise sites of binding were investigated along the cell cycle utilizing two novel procedures based on immunoprecipitation following UV irradiation with a pulsed laser light source. In G(1), the pre-replicative complex contains CDC6, MCM3, ORC1 and ORC2 proteins; of these, the post-replicative complex in S phase contains only ORC2; in M phase none of them are bound. The precise nucleotide of binding was identified for the two ORC and the CDC6 proteins near the start sites for leading-strand synthesis; the transition from the pre- to the post-replicative complex is accompanied by a 17 bp displacement of the ORC2 protein towards the start site.