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.

Enhancement of gene targeting in human cells by intranuclear permeation of the Saccharomyces cerevisiae Rad52 protein.

The introduction of exogenous DNA in human somatic cells results in a frequency of random integration at least 100-fold higher than gene targeting (GT), posing a seemingly insurmountable limitation for gene therapy applications. We previously reported that, in human cells, the stable over-expression of the Saccharomyces cerevisiae Rad52 gene (yRAD52), which plays the major role in yeast homologous recombination (HR), caused an up to 37-fold increase in the frequency of GT, indicating that yRAD52 interacts with the double-strand break repair pathway(s) of human cells favoring homologous integration. In the present study, we tested the effect of the yRad52 protein by delivering it directly to the human cells. To this purpose, we fused the yRAD52 cDNA to the arginine-rich domain of the TAT protein of HIV (tat11) that is known to permeate the cell membranes. We observed that a recombinant yRad52tat11 fusion protein produced in Escherichia coli, which maintains its ability to bind single-stranded DNA (ssDNA), enters the cells and the nuclei, where it is able to increase both intrachromosomal recombination and GT up to 63- and 50-fold, respectively. Moreover, the non-homologous plasmid DNA integration decreased by 4-fold. yRAD52tat11 proteins carrying point mutations in the ssDNA binding domain caused a lower or nil increase in recombination proficiency. Thus, the yRad52tat11 could be instrumental to increase GT in human cells and a 'protein delivery approach' offers a new tool for developing novel strategies for genome modification and gene therapy applications.

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.

Stimulation of the DNA unwinding activity of human DNA helicase II/Ku by phosphorylation.

The Ku autoantigen is a heterodimeric protein of 70- and 83-kDa subunits, endowed with duplex DNA end-binding capacity and DNA helicase activity (Human DNA Helicase II, HDH II). HDH II/Ku is well established as the DNA binding component, the regulatory subunit as well as a substrate for the DNA-dependent protein kinase DNA-PK, a complex involved in the repair of DNA double-strand breaks and in V(D)J recombination in eukaryotes. The effects of phosphorylation by this kinase on the helicase activity of Escherichia coli-produced HDH II/Ku were studied. The rate of DNA unwinding by recombinant HDH II/Ku heterodimer is stimulated at least fivefold upon phosphorylation by DNA-PK(cs). This stimulation is due to the effective transfer of phosphate residues to the helicase rather than the mere presence of the complex. In vitro dephosphorylation of HeLa cellular HDH II/Ku caused a significant decrease in the DNA helicase activity of this enzyme.

Modular structure of the human lamin B2 replicator.

The cis-acting elements necessary for the activity of DNA replication origins in metazoan cells are still poorly understood. Here we report a thorough characterization of the DNA sequence requirements of the origin associated with the human lamin B2 gene. A 1.2-kb DNA segment, comprising the start site of DNA replication and located within a large protein-bound region, as well as a CpG island, displays origin activity when moved to different ectopic positions. Genomic footprinting analysis of both the endogenous and the ectopic origins indicates that the large protein complex is assembled in both cases around the replication start site. Replacement of this footprinted region with an unrelated sequence, maintaining the CpG island intact, abolishes origin activity and the interaction with hORC2, a subunit of the origin recognition complex. Conversely, the replacement of 17 bp within the protected region reduces the extension of the protection without affecting the interaction with hORC2. This substitution does not abolish the origin activity but makes it more sensitive to the integration site. Finally, the nearby CpG island positively affects the efficiency of initiation. This analysis reveals the modular structure of the lamin B2 origin and supports the idea that sequence elements close to the replication start site play an important role in origin activation.

Analysis of the unwinding activity of the dimeric RECQ1 helicase in the presence of human replication protein A.

RecQ helicases are required for the maintenance of genome stability. Characterization of the substrate specificity and identification of the binding partners of the five human RecQ helicases are essential for understanding their function. In the present study, we have developed an efficient baculovirus expression system that allows us to obtain milligram quantities of recombinant RECQ1. Our gel filtration and dynamic light scattering experiments show that RECQ1 has an apparent molecular mass of 158 kDa and a hydrodynamic radius of 5.4 +/- 0.6 nm, suggesting that RECQ1 forms dimers in solution. The oligomeric state of RECQ1 remains unchanged upon binding to a single-stranded (ss)DNA fragment of 50 nt. We show that RECQ1 alone is able to unwind short DNA duplexes (<110 bp), whereas considerably longer substrates (501 bp) can be unwound only in the presence of human replication protein A (hRPA). The same experiments with Escherichia coli SSB show that RECQ1 is specifically stimulated by hRPA. However, hRPA does not affect the ssDNA-dependent ATPase activity of RECQ1. In addition, our far western, ELISA and co-immunoprecipitation experiments demonstrate that RECQ1 physically interacts with the 70 kDa subunit of hRPA and that this interaction is not mediated by DNA.

Human RECQ1 and RECQ4 helicases play distinct roles in DNA replication initiation.

Cellular and biochemical studies support a role for all five human RecQ helicases in DNA replication; however, their specific functions during this process are unclear. Here we investigate the in vivo association of the five human RecQ helicases with three well-characterized human replication origins. We show that only RECQ1 (also called RECQL or RECQL1) and RECQ4 (also called RECQL4) associate with replication origins in a cell cycle-regulated fashion in unperturbed cells. RECQ4 is recruited to origins at late G(1), after ORC and MCM complex assembly, while RECQ1 and additional RECQ4 are loaded at origins at the onset of S phase, when licensed origins begin firing. Both proteins are lost from origins after DNA replication initiation, indicating either disassembly or tracking with the newly formed replisome. Nascent-origin DNA synthesis and the frequency of origin firing are reduced after RECQ1 depletion and, to a greater extent, after RECQ4 depletion. Depletion of RECQ1, though not that of RECQ4, also suppresses replication fork rates in otherwise unperturbed cells. These results indicate that RECQ1 and RECQ4 are integral components of the human replication complex and play distinct roles in DNA replication initiation and replication fork progression in vivo.

Binding of DNA topoisomerases I and II to replication origins.

The interaction of DNA topology modifying enzymes with eukaryotic DNA replication origins can be detected with nucleotide precision exploiting the action of enzyme poisons specific for type I or type II DNA topoisomerases. Using the topoisomerase I poison camptothecin and the topoisomerase II poison VP16, the precise sites of interaction of these enzymes around the lamin B2 origin have been identified at different points in the cell cycle. The procedure can be applied to any origin for which the sequence has been identified within approximately 1 kb.

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.

Similia similibus: pairing of homologous chromosomes driven by the physicochemical properties of DNA.

Genetic recombination in eukaryotes requires the pairing of homologous chromosomes to allow precise molecular exchanges between chromosome pairs at intertwined structures called Holliday junctions, the formation of which requires the action of the RecA protein. The mechanism behind the precise pairing of structures as long as chromosomes remains mysterious. In yeast, during the initial phases of meiosis, chromosomes are paired at approximately 65 kilobase intervals via paranemic interactions that do not involve strand breakage nor the intervention of analogs of the RecA protein. It has been proposed that these paranemic interactions could occur between G-rich chromosomal regions, but putting in register stretches of homologous sequences hundreds of kb long remains challenging. Recent developments on the theory of the physicochemical properties of DNA in aqueous solutions, in presence of di- or multivalent counterions, leads to the prediction that molecules with the same sequence tend to pair spontaneously by paranemic interactions depending on the electrostatic properties of DNA. Experimental support for this prediction has now been provided in vitro with naked DNA. This newly discovered property of DNA duplexes may thus provide a clue to solve the puzzle of the premeiotic pairing.

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.

Is DNA sequence sufficient to specify DNA replication origins in metazoan cells?

DNA replication occupies a central position in the cell cycle and, therefore, in the development and life of multicellular organisms. During the last 10 years, our comprehension of this important process has considerably improved. Although the mechanisms that coordinate DNA replication with the other moments of the cell cycle are not yet fully understood, it is known that they mainly operate through DNA replication origins and the protein complexes bound to them. In eukaryotes, the packaging status of chromatin seems to be part of the mechanism that controls whether or not and when during the S-phase a particular origin will be activated. Intriguingly, the protein complexes bound to DNA replication origins appear to be directly involved in controlling chromatin packaging. In this manner they can also affect gene expression. In this review we focus on DNA replication origins in metazoan cells and on the relationship between these elements and the structural and functional organization of the genome.

Characterization of the DNA-unwinding activity of human RECQ1, a helicase specifically stimulated by human replication protein A.

The RecQ helicases are involved in several aspects of DNA metabolism. Five members of the RecQ family have been found in humans, but only two of them have been carefully characterized, BLM and WRN. In this work, we describe the enzymatic characterization of RECQ1. The helicase has 3' to 5' polarity, cannot start the unwinding from a blunt-ended terminus, and needs a 3'-single-stranded DNA tail longer than 10 nucleotides to open the substrate. However, it was also able to unwind a blunt-ended duplex DNA with a "bubble" of 25 nucleotides in the middle, as previously observed for WRN and BLM. We show that only short DNA duplexes (<30 bp) can be unwound by RECQ1 alone, but the addition of human replication protein A (hRPA) increases the processivity of the enzyme (>100 bp). Our studies done with Escherichia coli single-strand binding protein (SSB) indicate that the helicase activity of RECQ1 is specifically stimulated by hRPA. This finding suggests that RECQ1 and hRPA may interact also in vivo and function together in DNA metabolism. Comparison of the present results with previous studies on WRN and BLM provides novel insight into the role of the N- and C-terminal domains of these helicases in determining their substrate specificity and in their interaction with hRPA.

In vitro protein-DNA interactions at the human lamin B2 replication origin.

The complexity of mammalian origins of DNA replication has prevented, so far, the in vitro studies of the modalities of initiator protein binding and origin selection. We approached this problem by utilizing the human lamin B2 origin, wherein the precise start sites of replication initiation have been identified and known to be bound in vivo by the origin recognition complex (ORC). In order to analyze the in vitro interactions occurring at this origin, we have compared the DNA binding requirements and patterns of the human recombinant Orc4 with those of preparations of HeLa nuclear proteins containing the ORC complex. Here we show that both HsOrc4 alone and HeLa nuclear proteins recognize multiple sites within a 241-bp DNA sequence encompassing the lamin B2 origin. The DNA binding activity of HeLa cells requires the presence of ORC and can be reproduced in the absence of all the other proteins known to be recruited to origins by ORC. Both HsOrc4 alone and HeLa nuclear proteins exhibit cooperative and ATP-independent binding. This binding covers nucleotides 3853-3953 and then spreads outward. Because this region contains the start sites of DNA synthesis as well as the area protected in vivo and preserves protein binding capacity in vitro after removal of a fraction of the protected region, we suggest that it could contain the primary binding site. Thus the in vitro approach points to the sequence requirements for ORC binding as a key element for origin recognition.

Studies on the mode of Ku interaction with DNA.

The Ku heterodimer plays a central role in non-homologous end-joining. The binding of recombinant Ku to DNA has been investigated by dynamic light scattering, double-filter binding, fluorescence spectroscopy, and band shift assays. The hydrodynamic radius of Ku in solution is 5.2 nm and does not change when a 25-bp double-strand DNA (dsDNA) fragment (D25) is added, indicating that only one Ku molecule binds to a 25-bp fragment. The dissociation constant (k(d)) for the binding to D25 is 3.8 +/- 0.9 nm. If both ends of the substrate are closed with hairpin loops, Ku is still able to bind with little change in the k(d). The k(d) is not affected by ATP, Mg(2+), or ionic strength. However, the addition of bovine serum albumin decreases the k(d) by 2-fold. DNA substrates of 50 bp can bind two Ku molecules, whereas three molecules are bound to a 75-bp substrate. Data analysis with the Hill equation yields a value of the Hill coefficient (n) close to 1, and the k(d) values for the binding of Ku to both ends of these substrates are the same. Thus, we demonstrate that there is no cooperative interaction among the Ku heterodimers binding longer substrates.

Early mitotic degradation of the homeoprotein HOXC10 is potentially linked to cell cycle progression.

Hox proteins are transcription factors involved in controlling axial patterning, leukaemias and hereditary malformations. Here, we show that HOXC10 oscillates in abundance during the cell cycle, being targeted for degradation early in mitosis by the ubiquitin-dependent proteasome pathway. Among abdominal-B subfamily members, the mitotic proteolysis of HOXC10 appears unique, since the levels of the paralogous HOXD10 and the related homeoprotein HOXC13 are constant throughout the cell cycle. When two destruction box motifs (D-box) are mutated, HOXC10 is stabilized and cells accumulate in metaphase. HOXC10 appears to be a new prometaphase target of the anaphase-promoting complex (APC), since its degradation coincides with cyclin A destruction and is suppressed by expression of a dominant-negative form of UbcH10, an APC-associated ubiquitin-conjugating enzyme. Moreover, HOXC10 co-immunoprecipitates the APC subunit CDC27, and its in vitro degradation is reduced in APC-depleted extracts or by competition with the APC substrate cyclin A. These data imply that HOXC10 is a homeoprotein with the potential to influence mitotic progression, and might provide a link between developmental regulation and cell cycle control.