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.     

Defined sequence modules and an architectural element cooperate to promote initiation at an ectopic mammalian chromosomal replication origin

A small DNA fragment containing the high-frequency initiation region (IR) ori-beta from the hamster dihydrofolate reductase locus functions as an independent replicator in ectopic locations in both hamster and human cells. Conversely, a fragment of the human lamin B2 locus containing the previously mapped IR serves as an independent replicator at ectopic chromosomal sites in hamster cells. At least four defined sequence elements are specifically required for full activity of ectopic ori-beta in hamster cells. These include an AT-rich element, a 4-bp sequence located within the mapped IR, a region of intrinsically bent DNA located between these two elements, and a RIP60 protein binding site adjacent to the bent region. The ori-beta AT-rich element is critical for initiation activity in human, as well as hamster, cells and can be functionally substituted for by an AT-rich region from the human lamin B2 IR that differs in nucleotide sequence and length. Taken together, the results demonstrate that two mammalian replicators can be activated at ectopic sites in chromosomes of another mammal and lead us to speculate that they may share functionally related elements.     

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.     

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 origin recognition complex marks a replication origin in the human TOP1 gene promoter

The locations of the origin recognition complex (ORC) in mammalian genomes have been elusive. We have therefore analyzed the DNA sequences associated with human ORC via in vivo cross-linking and chromatin immunoprecipitation. Antibodies specific for hOrc2 protein precipitate chromatin fragments that also contain other ORC proteins, suggesting that the proteins form multisubunit complexes on chromatin in vivo. A binding region for ORC was identified at the CpG island upstream of the human TOP1 gene. Nascent strand abundance assays show that the ORC binding region coincides with an origin of bidirectional replication. The TOP1 gene includes two well characterized matrix attachment regions. The matrix attachment region elements analyzed contain no ORC and constitute no sites for replication initiation. In initial attempts to use the chromatin immunoprecipitation technique for the identification of additional ORC sites in the human genome, we isolated a sequence close to another actively transcribed gene (TOM1) and an alphoid satellite sequence that underlies centromeric heterochromatin. Nascent strand abundance assays gave no indication that the heterochromatin sequence serves as a replication initiation site, suggesting that an ORC on this site may perform functions other than replication initiation.     

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.     

Origin recognition complex binding to a metazoan replication origin

The initiation of DNA replication in eukaryotic cells at the onset of S phase requires the origin recognition complex (ORC) [1]. This six-subunit complex, first isolated in Saccharomyces cerevisiae [2], is evolutionarily conserved [1]. ORC participates in the formation of the prereplicative complex [3], which is necessary to establish replication competence. The ORC-DNA interaction is well established for autonomously replicating sequence (ARS) elements in yeast in which the ARS consensus sequence [4] (ACS) constitutes part of the ORC binding site [2, 5]. Little is known about the ORC-DNA interaction in metazoa. For the Drosophila chorion locus, it has been suggested that ORC binding is dispersed [6]. We have analyzed the amplification origin (ori) II/9A of the fly, Sciara coprophila. We identified a distinct 80-base pair (bp) ORC binding site and mapped the replication start site located adjacent to it. The binding of ORC to this 80-bp core region is ATP dependent and is necessary to establish further interaction with an additional 65-bp of DNA. This is the first time that both the ORC binding site and the replication start site have been identified in a metazoan amplification origin. Thus, our findings extend the paradigm from yeast ARS1 to multicellular eukaryotes, implicating ORC as a determinant of the position of replication initiation.     

Utilization of the same DNA replication origin by human cells of different derivation.

In the past, a highly sensitive and efficient method was developed to map DNA replication origins in human cells, based on quantitative PCR performed on nascent DNA samples. This method allowed the identification of a replication origin in the myeloid HL-60 cell line, located on chromosome 19 within an approximately 500 bp segment near the lamin B2 gene [Giacca et al. (1994) Proc. Natl. Acad. Sci. USA, 91, 7119]. The same procedure has now been further simplified and extended to a variety of other exponentially growing human cells of different histological derivation (three neural, one connectival and one epithelial), with a nearly diploid chromosomal content. In all the six cell lines tested, the origin activity within the lamin B2 gene domain was localized to the same region. Furthermore, the lamin B2 origin was also found to be active in stimulated, but not in quiescent, peripheral blood lymphocytes.     

New insights into replication origin characteristics in metazoans

We recently reported the identification and characterization of DNA replication origins (Oris) in metazoan cell lines. Here, we describe additional bioinformatic analyses showing that the previously identified GC-rich sequence elements form origin G-rich repeated elements (OGREs) that are present in 67% to 90% of the DNA replication origins from Drosophila to human cells, respectively. Our analyses also show that initiation of DNA synthesis takes place precisely at 160 bp (Drosophila) and 280 bp (mouse) from the OGRE. We also found that in most CpG islands, an OGRE is positioned in opposite orientation on each of the two DNA strands and detected two sites of initiation of DNA synthesis upstream or downstream of each OGRE. Conversely, Oris not associated with CpG islands have a single initiation site. OGRE density along chromosomes correlated with previously published replication timing data. Ori sequences centered on the OGRE are also predicted to have high intrinsic nucleosome occupancy. Finally, OGREs predict G-quadruplex structures at Oris that might be structural elements controlling the choice or activation of replication origins.     

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.     

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.     

Functional equivalency and diversity of cis-acting elements among yeast replication origins.

The DNA replication origins of the yeast Saccharomyces cerevisiae require several short functional elements, most of which are not conserved in sequence. To better characterize ARS305, a replicator from a chromosomal origin, we swapped functional DNA elements of ARS305 with defined elements of ARS1. ARS305 contains elements that are functionally exchangeable with ARS1 A and B1 elements, which are known to bind the origin recognition complex; however, the ARS1 A element differs in that it does not require a 3' box adjacent to the essential autonomously replicating sequence consensus. At the position corresponding to ARS1 B3, ARS305 has a novel element, B4, that can functionally substitute for every type of short element (B1, B2, and B3) in the B domain. Unexpectedly, the replacement of element B4 by ARS1 B3, which binds ABF1p and is known as a replication enhancer, inhibited ARS305 function. ARS305 has no short functional element at or near positions corresponding to the B2 elements in ARS1 and ARS307 but contains an easily unwound region whose functional importance was supported by a broad G+C-rich substitution mutation. Surprisingly, the easily unwound region can functionally substitute for the ARS1 B2 element, even though ARS1 B2 was found to possess a distinct DNA sequence requirement. The functionally conserved B2 element in ARS307 contains a known sequence requirement, and helical stability analysis of linker and minilinker mutations suggested that B2 also contains a DNA unwinding element (DUE). Our findings suggest that yeast replication origins employ a B2 element or a DUE to mediate a common function, DNA unwinding during initiation, although not necessarily through a common mechanism.     

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.     

cis-Acting components of human papillomavirus (HPV) DNA replication: linker substitution analysis of the HPV type 11 origin.

Papillomavirus DNA replication requires the viral trans-acting factors E1 and E2 in addition to the host cell's general replication machinery. The origins of DNA replication in bovine and human papillomavirus genomes have been localized to a specific part of the upstream regulatory region (URR) which includes recognition sites for E1 and E2 proteins. To fine map cis-acting elements influencing human papillomavirus type 11 (HPV-11) DNA replication and to determine the relative contributions of such sites, we engineered consecutive linker substitution mutations across a region of 158 bp in the HPV-11 origin and tested mutant origins for replication function in a cell-based transient replication assay. Our results both confirm and extend the findings of others. E2 binding sites are the major cis components of HPV-11 DNA replication, and there is evidence for synergy between these sites. Differential capacity of the three E2 binding sites within the origin to affect replication may be attributed, at least in part, to context. At least one E2 binding site is essential for replication. The imperfect AT-rich palindrome of the E1 helicase binding site is not essential since replication occurs even in the absence of this sequence. However, replication is enhanced by the presence of the palindromic sequence in the HPV-11 origin. Sequence components adjacent to the E1 and E2 binding sites, comprising AT-rich and purine-rich elements and the consensus TATA box sequence, probably contribute to the overall efficiency of replication, though they are nonessential. None of the other cis elements of the HPV-11 origin region analyzed seems to influence replication significantly in the system described. The HPV-11 origin of DNA replication therefore differs from those of the other papovaviruses, simian virus 40 and polyomavirus, inasmuch as an intact helicase binding site and adjacent AT-rich components, while influential, are not absolutely essential.     

Control of ATP-Dependent Binding of Saccharomyces cerevisiae Origin Recognition Complex to Autonomously Replicating DNA Sequences

Eukaryotic origin recognition complexes (ORCs) play pivotal roles in the initiation of chromosomal DNA replication. ORC from the yeast, Saccharomyces cerevisiae, recognizes and binds replication origins in the late G1 phase and the binding has profound implications in the progression of the cell cycle to the S-phase. Therefore, we have quantitatively analyzed the mechanism of recognition and interaction of the yeast ORC with various elements of a yeast origin of DNA replication, the autonomously replicating sequence 1 (ARS1). ORC bound all four individual A and B elements of ARS1 with reasonably high affinities. However, the highest affinity binding was observed with a DNA sequence containing both the A and B1 elements. In addition, ATP and ADP significantly modulated the binding of ORC to the combined elements as well as modulating the binding of ORC to the element A alone or in combination with the B1 element. However, binding of ORC to individual B1, B2, and B3 elements was not responsive to nucleotides. Thus, the consensus ARS sequence in element A appeared to play a pivotal role in the ATP-dependent binding of ORC to ARS1 and likely in other ARSs or origins of DNA replication.