DNA loop anchorage region colocalizes with the replication origin located downstream to the human gene encoding lamin B2

The recently developed procedure of topoisomerase II-mediated DNA loop excision has been used to analyze the topological organization of a human genome fragment containing the gene encoding lamin B2 and the ppv1 gene. A 3.5 kb long DNA loop anchorage/topoisomerase II cleavage region was found within the area under study. This region includes the end of the lamin B2 coding unit and an intergenic region where an origin of DNA replication was previously found. These observations further corroborate the hypothesis that DNA replication origins are located at or close to DNA loop anchorage regions. J. Cell. Biochem. 69:13–18, 1998. © 1998 Wiley-Liss, Inc.     

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.     

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.     

The quest for a humanori

Attempts at identifying DNA replication origins in human cells have been performed with a variety of molecular genetic and biochemical approaches, with often controversial results.The combination of bromodeoxyuridine labelling, immunopurification of newly synthesized labelled DNA, measurement of the relative abundance of markers in this DNA by quantitative competitive PCR, has allowed the identification within 450 bp of the start-site of DNA replication located at the human lamin B2 gene. The origin is located near the non-transcribed spacer between two highly transcribed genes and shows evidence of a number of specific protein-DNA interactions, the most prominent of which disappears when the cells are differentiated into a non-proliferating state.     

Mapping of an origin of DNA replication near the transcriptional promoter of the human HPRT gene

A quantitative PCR method was used to map a functional origin of DNA replication in the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene in normal human fibroblasts. This PCR method measures the abundance of specific sequences in short fragments of newly replicated DNA from logarithmically growing cells. Quantitative measurements rely on synthetic molecules (competitors) that amplify with the same primer sets as the target molecules, but generate products of different sizes. This method was first utilized to determine the position of the replication origin near the lamin B2 gene (Giacca et al. [1994] Proc. Natl. Acad. Sci. U S A. 91:7119-7123). In the present study, primer sets were tested along a 16-kb region near exon 1 of the HPRT gene. The most abundant fragment was found to be located in the first intron of HPRT, just downstream of the promoter and exon 1 of the gene, and approximately 3.5 kb upstream of a previously reported autonomously replicating sequence (Sykes et al. [1988] Mol. Gen. Genet. 212:301-309).     

High-throughput mapping of origins of replication in human cells

Mapping origins of replication has been challenging in higher eukaryotes. We have developed a rapid, genome-wide method to map origins of replication in asynchronous human cells by combining the nascent strand abundance assay with a highly tiled microarray platform, and we validated the technique by two independent assays. We applied this method to analyse the enrichment of nascent DNA in three 50-kb regions containing known origins of replication in the MYC, lamin B2 (LMNB2) and haemoglobin beta (HBB) genes, a 200-kb region containing the rare fragile site, FRAXA, and a 1,075-kb region on chromosome 22; we detected most of the known origins and also 28 new origins. Surprisingly, the 28 new origins were small in size and located predominantly within genes. Our study also showed a strong correlation between origin replication timing and chromatin acetylation.     

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 G0 resting cells, consistently with its involvement in origin function.     

One-way PCR-based mapping of a replication initiation point (RIP)

The lamin B2 locus is the only mammalian origin whose replication initiation points (RIPs) have been mapped. Although this paper was published 8 years ago, no further mammalian RIP-mapping studies have been reported, largely due to technical difficulties of ligation-mediated (LM)-PCR used by the authors. Here, we report the development of a simple, one-way PCR-based protocol that allows one to accurately determine RIPs at mammalian origins. The procedure can be completed within 48 h from the time of cell lysis in the agarose gel. Nascent DNA is then isolated from the same gel after DNA is separated by alkaline gel electrophoresis. Subsequently, RIPs are determined by one-way PCR-based primer extension using labeled primers. Using this protocol, we have successfully mapped RIPs in the human DBF4 locus. As one-way PCR is routinely used by many scientists, this protocol will provide a powerful new tool for studying DNA replication in many organisms including mammalian cells.     

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.     

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.     

Transient dsDNA breaks during pre-replication complex assembly

Initiation of DNA replication involves the ordered assembly of the multi-protein pre-replicative complex (pre-RC) during G₁ phase. Previously, DNA topoisomerase II (topo II) was shown to associate with the DNA replication origin located in the lamin B2 gene locus in a cell-cycle-modulated manner. Here we report that activation of both the early-firing lamin B2 and the late-firing hOrs8 human replication origins involves DNA topo II-dependent, transient, site-specific dsDNA-break formation. Topo IIβ in complex with the DNA repair protein Ku associates in vivo and in vitro with the pre-RC region, introducing dsDNA breaks in a biphasic manner, during early and mid-G₁ phase. Inhibition of topo II activity interferes with the pre-RC assembly resulting in prolonged G₁ phase. The data mechanistically link DNA topo IIβ-dependent dsDNA breaks and the components of the DNA repair machinery with the initiation of DNA replication and suggest an important role for DNA topology in origin activation.     

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 pre-replicative complex assembly and topoisomerase I allowing the topological adaptations necessary for initiation of bi-directional synthesis.     

Major DNA replication initiation sites in the c-myc locus in human cells

DNA replication initiation sites and initiation frequencies over 12. 5 kb of the human c-myc locus, including 4.6 kb of new 5' sequence, were determined based on short nascent DNA abundance measured by competitive polymerase chain reaction using 21 primer sets. In previous measurements, no comparative quantitation of nascent strand abundance was performed, and distinction of major from minor initiation sites was not feasible. Two major initiation sites were identified in this study. One predominant site has been located at approximately 0.5 kb upstream of exon 1 of the c-myc gene, and a second new major site is located in exon 2. The site in exon 2 has not been previously identified. In addition, there are other sites that may act as less frequently used initiation sites, some of which may correspond to sites in previous reports. Furthermore, a comparison of the abundance of DNA replication intermediates over this same region of the c-myc locus between HeLa and normal skin fibroblast (NSF) cells indicated that the relative distribution was very similar, but that nascent strand abundance in HeLa cells was approximately twice that in NSF relative to the abundance at the lamin B2 origin. This increased activity at initiation sites in the c-myc locus may mainly be influenced by regulators at higher levels in transformed cells like HeLa.     

Site-specific and temporally controlled initiation of DNA replication in a human cell-free system

We have recently established a cell-free system from human cells that initiates semi-conservative DNA replication in nuclei isolated from cells which are synchronised in late G₁ phase of the cell division cycle. We now investigate origin specificity of initiation using this system. New DNA replication foci are established upon incubation of late G₁ phase nuclei in a cytosolic extract from proliferating human cells. The intranuclear sites of replication foci initiated in vitro coincide with the sites of earliest replicating DNA sequences, where DNA replication had been initiated in these nuclei in vivo upon entry into S phase of the previous cell cycle. In contrast, intranuclear sites that replicate later in S phase in vivo do not initiate in vitro. DNA replication initiates in this cell-free system site-specifically at the lamin B2 DNA replication origin, which is also activated in vivo upon release of mimosine-arrested late G₁ phase cells into early S phase. In contrast, in the later replicating ribosomal DNA locus (rDNA) we neither detected replicating rDNA in the human in vitro initiation system nor upon entry of intact mimosine-arrested cells into S phase in vivo. As a control, replicating rDNA was detected in vivo after progression into mid S phase. These data indicate that early origin activity is faithfully recapitulated in the in vitro system and that late origins are not activated under these conditions, suggesting that early and late origins may be subject to different mechanisms of control.     

Noncanonical DNA elements in the lamin B2 origin of DNA replication

DNA replication origins of eukaryotes lack linear replicator elements but contain short (dT)(n) (dA)(n) sequences that could build mutually equivalent unorthodox structures. Here we report that the lamin B2 origin of DNA replication adopts an alternative form characterized by unpaired regions CTTTTTTTTTTCC/GGAAAAAAAAAAG (3900-3912) and CCTTTTTTTTC/GAAAAAAAAGG (4141-4151). Both unpaired regions are resistant to DNase and except in central parts of their homopyrimidine strands are sensitive to single strand-specific chemicals. Interactions that protect central pyrimidines probably stabilize the bubble-like areas. Because DNA fragments containing either one or both bubbles migrate in TBM (89 mm Tris base, 89 mm boric acid, and 2 mm MgCl(2)) PAGE even faster than expected from their linear size, interacting regions are expected to belong to the same molecule. In an origin fragment containing a single bubble, free homopyrimidine strand can only interact with Hoogsteen hydrogen bonding surfaces from a complementary double stranded sequence. Indeed, this origin fragment reacts with triplex preferring antibody. In competition binding experiments control double stranded DNA or single stranded (dT)(40) do not affect origin-antibody interaction, whereas TAT and GGC triplexes exert competitive effect. Because the chosen fragment does not contain potential GGC forming sequences, these experiments confirm that the lamin B2 origin adopts a structure partly composed of intramolecular TAT triads.     

Head and/or CaaX domain deletions of lamin proteins disrupt preformed lamin A and C but not lamin B structure in mammalian cells

The nuclear lamina is an important determinant of nuclear architecture. Mutations in A-type but not B-type lamins cause a range of human genetic disorders, including muscular dystrophy. Dominant mutations in nuclear lamin proteins have been shown to disrupt a preformed lamina structure in Xenopus egg extracts. Here, a series of deletion mutations in lamins A and B1 were evaluated for their ability to disrupt lamina structure in Chinese hamster ovary cells. Deletions of either the lamin A "head" domain or the C-terminal CaaX domain formed intranuclear aggregates and resulted in the disruption of endogenous lamins A/C but not lamins B1/B2. By contrast, "head-less" lamin B1 localized to the nuclear rim with no detectable effect on endogenous lamins, whereas lamin B1 CaaX domain deletions formed intranuclear aggregates, disrupting endogenous lamins A/C but not lamins B1/B2. Filter binding assays revealed that a head/CaaX domain lamin B1 mutant interacted much more strongly with lamins A/C than with lamins B1/B2. Regulated induction of this mutant in stable cell lines resulted in the rapid elimination of all detectable lamin A protein, whereas lamin C was trapped in a soluble form within the intranuclear aggregates. In contrast to results in Xenopus egg extracts, dominant negative lamin B1 (but not lamin A) mutants trapped replication proteins involved in both the initiation and elongation phases of replication but did not effect cellular growth rates or the assembly of active replication centers. We conclude that elimination of the CaaX domain in lamin B1 and elimination of either the CaaX or head domain in lamin A constitute dominant mutations that can disrupt A-type but not B-type lamins, highlighting important differences in the way that A- and B-type lamins are integrated into the lamina.     

The histone deacetylase inhibitor trichostatin A alters the pattern of DNA replication origin activity in human cells

Eukaryotic chromatin structure limits the initiation of DNA replication spatially to chromosomal origin zones and temporally to the ordered firing of origins during S phase. Here, we show that the level of histone H4 acetylation correlates with the frequency of replication initiation as measured by the abundance of short nascent DNA strands within the human c-myc and lamin B2 origins, but less well with the frequency of initiation across the β-globin locus. Treatment of HeLa cells with trichostatin A (TSA) reversibly increased the acetylation level of histone H4 globally and at these initiation sites. At all three origins, TSA treatment transiently promoted a more dispersive pattern of initiations, decreasing the abundance of nascent DNA at previously preferred initiation sites while increasing the nascent strand abundance at lower frequency genomic initiation sites. When cells arrested in late G₁ were released into TSA, they completed S phase more rapidly than untreated cells, possibly due to the earlier initiation from late-firing origins, as exemplified by the β-globin origin. Thus, TSA may modulate replication origin activity through its effects on chromatin structure, by changing the selection of initiation sites, and by advancing the time at which DNA synthesis can begin at some initiation sites.