The ease of DNA unwinding as a determinant of initiation at yeast replication origins

doi:10.1016/0092-8674(88)90469-2

Cell

Volume 52, Issue 4, 26 February 1988, Pages 559-567

https://doi.org/10.1016/0092-8674(88)90469-2 Get rights and content

Abstract

We have localized the DNA sequence that facilitates unwinding of a yeast replication origin, the H4 ARS. The readily unwound sequence lies adjacent to the previously characterized consensus core sequence of the ARS. Unwinding is detected through the formation of a single-strand-specific nuclease hypersensitive site in H4 ARS mutant derivatives present on super-coiled plasmids. Linker-scanning and linker-deletion derivatives exhibit wild-type nuclease hypersensitivity and ARS function, while large external deletions reduce or eliminate nuclease detectable unwinding and origin function. ARS unwinding and origin function can be rescued in the deletion mutants by inserting a biologically unrelated sequence with DNA unwinding properties similar to a functional ARS. The data clarify the nature of DNA sequence requirements in the ARS by suggesting that small substitutions, insertions, and deletions are tolerated in the region flanking the consensus core sequence because they do not significantly alter the unwinding properties of the region.

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Structure, replication efficiency and fragility of yeast ARS elements
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Citation Excerpt :
It was proposed that the multiple sequences in the vicinity of ACSs enable cooperative binding of replication initiation proteins (Theis and Newlon, 1994). The sequences of the supplemental regions do not have a conserved sequence motif but are A + T-rich, lowering the free energy of unwinding of the DNA helix to facilitate the association of initiation proteins (Umek and Kowalski, 1988). ARS function relies on the ease of unwinding of T-rich region 3′ to the consensus sequence.
DNA replication in eukaryotes initiates at specific sites known as origins of replication, or replicators. These replication origins occur throughout the genome, though the propensity of their occurrence depends on the type of organism. In eukaryotes, zones of initiation of replication spanning from about 100 to 50,000 base pairs have been reported. The characteristics of eukaryotic replication origins are best understood in the budding yeast Saccharomyces cerevisiae, where some autonomously replicating sequences, or ARS elements, confer origin activity. ARS elements are short DNA sequences of a few hundred base pairs, identified by their efficiency at initiating a replication event when cloned in a plasmid. ARS elements, although structurally diverse, maintain a basic structure composed of three domains, A, B and C. Domain A is comprised of a consensus sequence designated ACS (ARS consensus sequence), while the B domain has the DNA unwinding element and the C domain is important for DNA-protein interactions. Although there are ∼400 ARS elements in the yeast genome, not all of them are active origins of replication. Different groups within the genus Saccharomyces have ARS elements as components of replication origin. The present paper provides a comprehensive review of various aspects of ARSs, starting from their structural conservation to sequence thermodynamics. All significant and conserved functional sequence motifs within different types of ARS elements have been extensively described. Issues like silencing at ARSs, their inherent fragility and factors governing their replication efficiency have also been addressed. Progress in understanding crucial components associated with the replication machinery and timing at these ARS elements is discussed in the section entitled “The replicon revisited”.
DNA A-tracts bending: Polarization effects on electrostatic interactions across their minor groove
2008, Journal of Theoretical Biology
Citation Excerpt :
DNA bending associated with the A-tracts, runs of four to six consecutive adenine residues in phase with the helical repeat, has generated considerable interest for the past quarter of a century. This interest has been stimulated by their excessive concentration in the promoters of a range of eukaryotic and prokaryotic species (Ross et al., 2001; Shomer and Yagil, 1999; Yagil, 2006) and their participation in the folding of DNA in the nucleosome (Satchwell et al., 1986), as well as by their presence within the replication origins (Bramhill and Kornberg, 1988; Gille and Messer, 1991; Umek and Kowalski, 1988). The anomalous electrophoretic mobility of the A-tracts (Diekmann, 1987; Koo et al., 1986; Marini et al., 1982; Zinkel and Crothers, 1987) was found to be consistent with bending toward their minor groove (Zinkel and Crothers, 1987).
Bending by the DNA A-tracts constitutes a contentious issue, suggesting deficiencies in the physics employed so far. Here, we inquire as to the importance in this bending of many-body polarization effects on the electrostatic interactions across their narrow minor groove. We have done this on the basis of the findings of Jarque and Buckingham who developed a procedure based on a Monte Carlo simulation for two charges of the same sign embedded in a polarizable medium. Remarkably, the present analysis reveals that for compact DNA conformations, which result from dynamic effects, an overall attractive interaction operates between the phosphate charges; this interaction is especially strong for the narrow minor groove of the A-tracts, suggesting a tendency for DNA to bend toward this groove. This tendency is in agreement with the conclusions of electrophoretic and NMR solution studies. The present analysis is also consistent with the experimental observations that the minor groove is much more easily compressible than the major groove and the bending propensity of the A-tracts is greatly reduced at “premelting” temperatures. By contrast, the dielectric screening model predicts a repulsion between the phosphate charges and is not consistent with the aforementioned bending tendency or experimental observations.
Structure and function of the c-myc DNA-unwinding element-binding protein DUE-B
2007, Journal of Biological Chemistry
Local zones of easily unwound DNA are characteristic of prokaryotic and eukaryotic replication origins. The DNA-unwinding element of the human c-myc replication origin is essential for replicator activity and is a target of the DNA-unwinding element-binding protein DUE-B in vivo. We present here the 2.0Å crystal structure of DUE-B and complementary biochemical characterization of its biological activity. The structure corresponds to a dimer of the N-terminal domain of the full-length protein and contains many of the structural elements of the nucleotide binding fold. A single magnesium ion resides in the putative active site cavity, which could serve to facilitate ATP hydrolytic activity of this protein. The structure also demonstrates a notable similarity to those of tRNA-editing enzymes. Consistent with this structural homology, the N-terminal core of DUE-B is shown to display both d-aminoacyl-tRNA deacylase activity and ATPase activity. We further demonstrate that the C-terminal portion of the enzyme is disordered and not essential for dimerization. However, this region is essential for DNA binding in vitro and becomes ordered in the presence of DNA.
Binding of the universal minicircle sequence binding protein at the kinetoplast DNA replication origin
2006, Journal of Biological Chemistry
Kinetoplast DNA, the mitochondrial DNA of trypanosomatids, is a remarkable DNA structure that contains, in the species Crithidia fasciculata, 5000 topologically linked duplex DNA minicircles. Their replication initiates at two conserved sequences, a dodecamer, known as the universal minicircle sequence (UMS), and a hexamer, which are located at the replication origins of the minicircle L and H strands, respectively. A UMS-binding protein (UMSBP) binds specifically the 12-mer UMS sequence and a 14-mer sequence that contains the conserved hexamer in their single-stranded DNA conformation. In vivo cross-linking analyses reveal the binding of UMSBP to kinetoplast DNA networks in the cell. Furthermore, UMSBP binds in vitro to native minicircle origin fragments, carrying the UMSBP recognition sequences. UMSBP binding at the replication origin induces conformational changes in the bound DNA through its folding, aggregation and condensation.
Chromatin structural elements and chromosomal translocations in leukemia
2006, DNA Repair
Recurring chromosome abnormalities are strongly associated with certain subtypes of leukemia, lymphoma and sarcomas. More recently, their potential involvement in carcinomas, i.e. prostate cancer, has been recognized. They are among the most important factors in determining disease prognosis, and in many cases, identification of these chromosome abnormalities is crucial in selecting appropriate treatment protocols. Chromosome translocations are frequently observed in both de novo and therapy-related acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). The mechanisms that result in such chromosome translocations in leukemia and other cancers are largely unknown. Genomic breakpoints in all the common chromosome translocations in leukemia, including t(4;11), t(9;11), t(8;21), inv(16), t(15;17), t(12;21), t(1;19) and t(9;22), have been cloned. Genomic breakpoints tend to cluster in certain intronic regions of the relevant genes including MLL, AF4, AF9, AML1, ETO, CBFB, MYHI1, PML, RARA, TEL, E2A, PBX1, BCR and ABL. However, whereas the genomic breakpoints in MLL tend to cluster in the 5′ portion of the 8.3 kb breakpoint cluster region (BCR) in de novo and adult patients and in the 3′ portion in infant leukemia patients and t-AML patients, those in both the AML1 and ETO genes occur in the same clustered regions in both de novo and t-AML patients. These differences may reflect differences in the mechanisms involved in the formation of the translocations. Specific chromatin structural elements, such as in vivo topoisomerase II (topo II) cleavage sites, DNase I hypersensitive sites and scaffold attachment regions (SARs) have been mapped in the breakpoint regions of the relevant genes. Strong in vivo topo II cleavage sites and DNase I hypersensitive sites often co-localize with each other and also with many of the BCRs in most of these genes, whereas SARs are associated with BCRs in MLL, AF4, AF9, AML1, ETO and ABL, but not in the BCR gene. In addition, the BCRs in MLL, AML1 and ETO have the lowest free energy level for unwinding double strand DNA. Virtually all chromosome translocations in leukemia that have been analyzed to date show no consistent homologous sequences at the breakpoints, whereas a strong non-homologous end joining (NHEJ) repair signature exists at all of these chromosome translocation breakpoint junctions; this includes small deletions and duplications in each breakpoint, and micro-homologies and non-template insertions at genomic junctions of each chromosome translocation. Surprisingly, the size of these deletions and duplications in the same translocation is much larger in de novo leukemia than in therapy-related leukemia. We propose a non-homologous chromosome recombination model as one of the mechanisms that results in chromosome translocations in leukemia. The topo II cleavage sites at open chromatin regions (DNase I hypersensitive sites), SARs or the regions with low energy level are vulnerable to certain genotoxic or other agents and become the initial breakage sites, which are followed by an excision end joining repair process.
The c-myc DNA-unwinding element-binding protein modulates the assembly of DNA replication complexes in vitro
2005, Journal of Biological Chemistry
The presence of DNA-unwinding elements (DUEs) at eukaryotic replicators has raised the question of whether these elements contribute to origin activity by their intrinsic helical instability, as protein-binding sites, or both. We used the human c-myc DUE as bait in a yeast one-hybrid screen and identified a DUE-binding protein, designated DUE-B, with a predicted mass of 23.4 kDa. Based on homology to yeast proteins, DUE-B was previously classified as an aminoacyl-tRNA synthetase; however, the human protein is ∼60 amino acids longer than its orthologs in yeast and worms and is primarily nuclear. In vivo, chromatin-bound DUE-B localized to the c-myc DUE region. DUE-B levels were constant during the cell cycle, although the protein was preferentially phosphorylated in cells arrested early in S phase. Inhibition of DUE-B protein expression slowed HeLa cell cycle progression from G₁ to S phase and induced cell death. DUE-B extracted from HeLa cells or expressed from baculovirus migrated as a dimer during gel filtration and co-purified with ATPase activity. In contrast to endogenous DUE-B, baculovirus-expressed DUE-B efficiently formed high molecular mass complexes in Xenopus egg and HeLa extracts. In Xenopus extracts, baculovirus-expressed DUE-B inhibited chromatin replication and replication protein A loading in the presence of endogenous DUE-B, suggesting that differential covalent modification of these proteins can alter their effect on replication. Recombinant DUE-B expressed in HeLa cells restored replication activity to egg extracts immunodepleted with anti-DUE-B antibody, suggesting that DUE-B plays an important role in replication in vivo.

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Cell

ArticleThe ease of DNA unwinding as a determinant of initiation at yeast replication origins

Abstract

J. Biol. Chem.

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Fine-structure analysis of the DNA sequence requirements for autonomous replication of Saccharomyces cerevisiae

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Localization and sequence analysis of yeast origins of DNA replication

Deletion mutations affecting autonomously replicating sequence ARS1 of Saccharomyces cerevisiae

Mol. Cell Biol.

Simian virus 40 (SV40) DNA replication. SV40 large T antigen unwinds DNA containing the SV40 origin of replication

Specialized nucleoprotein structures at the origin of replication of bacteriophage lambda localized unwinding of duplex DNA by a six protein reaction

Article
The ease of DNA unwinding as a determinant of initiation at yeast replication origins