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Thermal Energy Suppresses Mutational Defects in DNA

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Thermal Energy Suppresses Mutational Defects in DNA Proc. Natl. Acad. Sci. USA Vol. 87, pp. 2486-2490, April 1990 Biochemistry Thermal energy suppresses mutational defects in DNA unwinding at a yeast replication origin (DNA replication/autonomously replicating sequences/Saccharomyces cerevisiae/supercoiled DNA/DNA unwinding element) ROBERT M. UMEK* AND DAVID KOWALSKIt Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, NY 14263 Communicated by Martin Gellert, January 18, 1990 (received for review November 30, 1989) ABSTRACT Yeast replication origins contain a DNA se- Here we directly demonstrate that the nuclease-hyper- quence element whose biological activity correlates with hy- sensitive element in the H4 ARS has a low free-energy persensitivity to single-strand-specific nucleases in negatively requirement for DNA unwinding and, furthermore, that supercoiled plasmids. By using two-dimensional gel electro- thermal energy facilitates origin unwinding in vitro. These phoresis of plasmid topoisomers, we demonstrate that thermo- findings, combined with the fact that the unwinding free dynamically stable origin unwinding accounts for the nuclease energies ofdifferent nuclease-hypersensitive elements can be hypersensitivity and, furthermore, that increased thermal en- ranked by their position in the hierarchy of nuclease hyper- ergy facilitates stable origin unwinding in vitro. In living cells, sensitivity (13), provided an experimental rationale for test- increased thermal energy can suppress orin mutations that ing whether the energy cost for unwinding the ARS 3' raise the free-energy cost for unwinding the nuclease- flanking sequence is important for biological function. We hypersensitive element. Specifically, mutational defects in au- report that mutations in the nuclease-hypersensitive element tonomously replicating sequence (ARS)-mediated plasmid rep- that sufficiently increase the free-energy cost for origin lication are less severe in cells grown at 300C as compared to unwinding lead to an enhanced dependence on elevated 230C. Our findings indicate that the energetics of DNA un- temperature for efficient plasmid replication. The data winding at the nuclease-hypersensitive element are biologically strongly support the hypothesis that the nuclease-hypersen- important. We call the nuclease-hypersensitive sequence the sitive element facilitates localized unwinding at the replica- DNA unwinding element (DUE) and propose that it serves as tion origin in yeast cells. We call the nuclease-hypersensitive into the DNA helix. sequence the DNA unwinding element (DUE) and propose the entry site for yeast replication enzymes that, like the DUE in the Escherichia coli chromosomal origin (14), it serves as the entry site for replication enzymes into the Origins ofDNA replication, through a combination ofgenetic DNA helix. and physical mapping techniques, have been identified in the yeast Saccharomyces cerevisiae (1-4). The origins were originally defined as autonomously replicating sequences MATERIALS AND METHODS (ARSs). The DNA sequence requirements of ARS function Enzymes. P1 nuclease, prepared according to Fujimoto et have been studied through comparative and mutational se- al. (15), was from Yamasa Shoyu (Choshi, Japan). T4 DNA quence analysis (5-7). An 11-base-pair core consensus se- ligase was from New England Biolabs and calf thymus DNA quence [(A/T)TTTAT(A/G)TTT(A/T)] is required for origin topoisomerase I was from Bethesda Research Laboratories. function. The core consensus sequence is thought to be the DNA. All plasmids were grown in E. coli HB101 cells in recognition site for an initiator protein, since point mutations Luria-Bertani broth. DNA was obtained from cells lysed by in the sequence lead to loss ofARS function (6). Additionally, boiling in the presence of lysozyme (16). The extractable a broad flanking sequence, 3' to the core consensus, is also superhelical density of different plasmids prepared by this required for efficient replication (7). Sequence comparisons procedure is -0.067, as determined by agarose gel electro- and mutational analyses have been less revealing about the phoresis (17) in the presence of chloroquine. The DNA was role of the flanking sequence. Although high in A+T content purified by two rounds of equilibrium centrifugation in ce- in general, the flanking sequence exhibits little primary sium chloride density gradients containing ethidium bromide sequence homology from one ARS to another (5). Further- (18). more, the flanking sequence tolerates certain substitutions, Preparation of Plasmid Topoisomers and Two-Dimensional insertions, and deletions within the required region (7, 8). A Gel Electrophoresis. Plasmid topoisomers were prepared us- variety of roles have been proposed for the flanking DNA ing a modification of the protocol presented by Wang (19). sequence (for review, see refs. 9 and 10); however, the actual Supercoiled pAB9 (8) was incubated with P1 nuclease and role(s) of the ARS 3' flanking sequence has not been firmly quantitatively converted to nicked-circular DNA (20). The established. nicks were then covalently closed using T4 DNA ligase in the has demonstrated that the 3' flanking presence of various concentrations of ethidium bromide. Our laboratory Twelve separate ligation reactions (containing ethidium bro- sequence of ARSs is hypersensitive to single-strand-specific mide concentrations from 1.2 to 7.8 tkg/ml) were pooled to endonucleases in negatively supercoiled DNA (11, 12). We generate the topoisomer population shown in Fig. 1A. Alter- presumed that the nuclease hypersensitivity reflects a low natively, the entire spectrum of topoisomers was generated free-energy requirement for unwinding. We proposed that the by limited topoisomerase I relaxation of supercoiled mole- ease of unwinding the origin is a determinant of replication extracted from E. coli. Gel of initiation since the nuclease hypersensitivity of origin mu- cules (21) electrophoresis tants correlates with autonomous replication (12). Abbreviations: DUE, DNA unwinding element; ARS, autonomously replicating sequence. The publication costs of this article were defrayed in part by page charge *Present address: Carnegie Institution of Washington, Department payment. This article must therefore be hereby marked "advertisement" of Embryology, Baltimore, MD 21210. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 2486 Downloaded by guest on September 28, 2021 Biochemistry: Umek and Kowalski Proc. Natl. Acad. Sci. USA 87 (1990) 2487 plasmid topoisomers was performed in submerged 1% agar- replication origin, pAB9 topoisomers were incubated with ose gels. Topoisomers were separated in the first dimension the single-strand-specific nuclease P1 prior to two-dimen- at 230C or 370C in the same solution conditions used for the sional gel electrophoresis. The results are shown in Fig. 1B. P1 nuclease reaction (12). A 1-cm-wide strip containing the The stably unwound topoisomers are preferentially cleaved topoisomers separated in the first dimension was fused to a and are converted to the nicked circular form (compare to second-dimension gel. The second-dimension gel was equil- Fig. 1A). Mapping of the P1 nuclease cleavages (data not ibrated to 36 mM Tris/30 mM NaH2PO4/1 mM Na2EDTA, shown) reveals that the yeast origin sequence is hypersensi- pH 7.8, containing chloroquine (5 pug/ml). After second- tive in the stably unwound topoisomers. Therefore, thermo- dimension electrophoresis, the gels were soaked in distilled dynamically stable DNA unwinding accounts for the nucle- water to remove the chloroquine, then soaked in distilled ase hypersensitivity of the yeast replication origin. We con- water containing ethidium bromide (1 ,ug/ml), and photo- clude that the nuclease-hypersensitive element has a low graphed under UV illumination. free-energy requirement for localized DNA unwinding rela- Yeast Transformations. The S. cerevisiae strain used to test tive to other sequences on the plasmid. plasmids for autonomous replication, YPH3 (ura3-52, lys- Thermal Energy Facilitates Stable Origin Unwinding. A+T- 801, ade2-101), was obtained from Philip Hieter (Johns rich sequences that are nuclease hypersensitive on super- Hopkins University). The test plasmids were transfected into coiled plasmids at 370C do not display this property at lower the recipient cells using the lithium acetate procedure orig- temperature (26). We wished to determine whether stable inally described by Ito et al. (22) and modified by Boguslaw- unwinding at the A+T-rich flanking sequence in a yeast ski (23). Aliquots ofthe transformed cells were plated at 230C replication origin is also temperature dependent. Topoiso- or 30°C onto synthetic minimal medium containing adenine mers of pAB9 were subjected to two-dimensional gel elec- sulfate (20 ,ug/ml) and lysine hydrochloride (30 ,ug/ml). The trophoresis as above except that the first-dimension electro- transformation frequency was -3000 transformants per,g of phoresis was carried out at 23TC instead of 370C. The results plasmid DNA. The selective liquid medium used for cell are shown in Fig. 1C. Each topoisomer migrates faster than doubling time experiments was the same as that used in the topoisomers offewer supercoils in the first dimension (top to selective plates. bottom). Thus, stable DNA unwinding is not detectable in the pAB9 plasmid at 230C in the range of superhelicity examined. RESULTS AND DISCUSSION Combined with the results presented in Fig. 1 A and B,
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