We have discovered that DNA supercoiling, in the absence of replication proteins, induces localized unwinding in the Escherichia coli replication origin (oriC) at the same sequence opened by the dnaA initiator protein. The DNA helix at the tandemly repeated, 13mer sequence is thermodynamically unstable, as evidenced by hypersensitivity to single‐strand‐specific nuclease in a negatively supercoiled plasmid, and demonstrated by stable DNA unwinding seen after two‐dimensional gel electrophoresis of topoisomers. A replication‐defective oriC mutant lacking the leftmost 13mer shows no nuclease hypersensitivity in two remaining 13mers and no detectable DNA unwinding on two‐dimensional gels. The replication defect in the oriC mutant can be corrected by inserting a dissimilar DNA sequence with reduced helical stability in place of the leftmost 13mer. Thus, the helical instability of the leftmost 13mer, not the specific 13mer sequence, is essential for origin function. The rightmost 13mer exhibits helical instability but differs from the leftmost 13mer in its strict sequence conservation among related bacterial origins. The repeated 13mer region appears to serve two overlapping functions: protein recognition and helical instability. We propose that the cis‐acting sequence whose helical instability is required for origin function be called the DNA unwinding element (DUE).

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A long A+T-rich sequence in supercoiled pBR322 DNA is hypersensitive to single-strand-specific nucleases at 370C but not at reduced temperature. The basis for the nuclease hypersensitivity is stable DNA unwinding as revealed by (i) the same temperature dependence for hypersensitivity and for stable unwinding of plasmid topoisomers after two-dimensional gel electrophoresis, (ii) preferential nuclease digestion of stably unwound topoisomers, and (iti) quantitative nicking of stably unwound topoisomers in the A+T-rich region. Nuclease hypersensitivity of A+T-rich sequences is hierarchical, and either deletion of the primary site or a sufficient increase in the free energy of supercoiling leads to enhanced nicking at An alternative A+T-rich site. ThM hierarchy of nuclease hypersensitivity reflects a hierarchy in the free energy required for unwinding naturally occurring sequences in supercoiled DNA. This finding, along with the known hypersensitivity of replication origins and transcriptional regulatory regions, has important implications for using single-strand-specific nucleases in DNA structure-function studies.Negatively supercoiled DNA is known to form stably unwound DNA conformations including Z-DNA, cruciform, and homopurine-homopyrimidine structures. Synthetic DNAs consisting of simple repeated sequences or long inverted repeat sequences cloned into plasmids have been useful for assessing the occurrence and energetics of stable DNA unwinding associated with these structures by twodimensional gel electrophoresis of topoisomers (1-6).In contrast to the synthetic DNAs studied, naturally occurring DNA sequences present in plasmids or viral (phage) genomes are primarily heterogeneous. The energetics of unwinding naturally occurring DNA sequences is of biological importance since DNA must unwind to initiate replication and transcription. However, stable DNA unwinding has not been detected in naturally occurring sequences present in supercoiled pBR322 and related plasmids under the same conditions where synthetic DNAs inserted into these plasmids readily unwind (1-6). Clearly, additional approaches are needed to identify naturally occurring sequences with a low free energy for unwinding in supercoiled DNA.We have been using single-strand-specific nucleases, especially mung bean nuclease and P1 nuclease, to probe for naturally occurring DNA sequences that unwind in plasmids and viral genomes (7-10). In supercoiled DNA, one, or a small subset, of A+T-rich sequences present in a plasmid or viral genome are nuclease hypersensitive under certain neutral pH conditions where sequences that form cruciforms, Z-DNA, and homopurine-homopyrimidine structures are not cleaved (5,6,(8)(9)(10). The hypersensitive sites are cleaved as much as 28,000 times faster than the nuclease-sensitive sites present in relaxed DNA (11). The identity of the nuclease hypersensitive sites could not be predicted based simply on A + T content (10) or early melting behavior (11). The hypersensitive sites are of potential biological significan...

SUMMARY DNA damage that blocks replication is bypassed in order to complete chromosome duplication and preserve cell viability and genome stability. Rad5, a PCNA polyubiquitin ligase and DNA-dependent ATPase in yeast, is orthologous to putative tumor suppressors and controls error-free damage bypass by an unknown mechanism. To identify the mechanism in vivo, we investigated the roles of Rad5 and analyzed the DNA structures that form during damage bypass at site-specific stalled forks present at replication origins. Rad5 mediated the formation of recombination-dependent, X-shaped DNA structures containing Holliday junctions between sister chromatids. Mutants lacking these damage-induced chromatid junctions were defective in resolving stalled forks, restarting replication and completing chromosome duplication. Rad5 polyubiquitin ligase and ATPase domains both contributed to replication fork recombination. Our results indicate that multiple activities of Rad5 function coordinately with homologous recombination factors to enable replication template switch events that join sister chromatids at stalled forks and bypass DNA damage.

We have defined a replication origin, ORI305, within chromosome III of Saccharomyces cerevisiae by means of mutational analysis. cis‐acting elements required for origin activity in the chromosome, as assayed by two‐dimensional gel electrophoresis of replication intermediates, are the same as those required for the function of an autonomously replicating sequence, ARS305, in a plasmid. Essential elements include (i) an 11 bp sequence that is a near match to the ARS consensus and (ii) a broad sequence directly 3′ to the consensus near match. Origin function is inactivated by point mutations in the essential near match sequence, suggesting that the sequence contributes to specifying the origin in the chromosome. Other consensus near matches with different sequences are present but are not required. The essential 3′‐flanking sequence exhibits DNA helical instability and is sensitive to deletion mutations that stabilize the DNA helix. The wild‐type 3′‐flanking sequence can be functionally substituted by dissimilar sequences that also exhibit helical instability. The requirement for DNA helical instability indicates that the essential 3′‐flanking sequence serves as a DNA unwinding element in the chromosome.