Effect of deletion and insertion on double-strand-break repair in Saccharomyces cerevisiae.

I investigated double-strand-break repair in Saccharomyces cerevisiae cells by measuring the frequencies and types of integration events at the PET56-HIS3-DED1 chromosomal region associated with the introduction of linearized plasmid DNAs containing homologous sequences. In general, the integration frequencies observed in strains containing a wild-type region, a 1-kilobase (kb) deletion, or a 5-kb insertion were similar, provided that the cleavage site in the plasmid DNA was present in the host genome. Cleavage at a plasmid DNA site corresponding to a region deleted in the chromosome caused a 10-fold reduction in the integration frequency even when the site was close to regions of homology. However, although the integration frequency was normal even when cleavage occurred only 25 base pairs (bp) outside the deletion breakpoint, 98% of the events were associated not with the usual heterogenote structure, but instead with a homogenote structure containing two copies of the deletion allele separated by vector sequences. Similarly, when cleavage occurred 80 bp outside the 5-kb substitution breakpoint, 40% of the integration events were associated with homogenote structures. From these observations, I suggest that exonuclease and polymerase activities are not rate-limiting steps in double-strand-break repair, exonuclease activity is coupled to the initiation step, the integration frequency is strongly influenced by the amount of homology near the recombinogenic ends, both ends of a linear DNA molecule might interact with the host chromosome before significant exonuclease or polymerase action, and the average repair tract is about 600 bp.     

Defining the consensus sequences of E.coli promoter elements by random selection.

The consensus sequence of E.coli promoter elements was determined by the method of random selection. A large collection of hybrid molecules was produced in which random-sequence oligonucleotides were cloned in place of a wild-type promoter element, and functional -10 and -35 E.coli promoter elements were obtained by a genetic selection involving the expression of a structural gene. The DNA sequences and relative levels of function for -10 and -35 elements were determined. The consensus sequences determined by this approach are very similar to those determined by comparing DNA sequences of naturally occurring E.coli promoters. However, no strong correlation is observed between similarity to the consensus and relative level of function. The results are considered in terms of E.coli promoter function and of the general applicability of the random selection method.     

Transcription of the his3 gene region in Saccharomyces cerevisiae

The dodecamer d(CpGpCpGpApApTpTpCpGpCpG) or C-G-C-G-A-A-T-T-C-G-C-G crystallizes as slightly more than one full turn of right-handed B-DNA. It is surrounded in the crystal by one bound spermine molecule and 72 ordered water molecules, most of which associate with polar N and O atoms at the exposed edges of base-pairs. Hydration within the major groove is principally confined to a monolayer of water molecules associated with exposed N and O groups on the bases, with most association being monodentate. Waters hydrating backbone phosphate oxygens tend not to be ordered, except where they are immobilized by 5-methyl groups from nearby thymines. In contrast, the minor groove is hydrated in an extensive and regular manner, with a zigzag “spine” of first- and second-shell hydration along the floor of the groove serving as a foundation for less-regular outer shells extending beyond the radius of the phosphate backbone. This spine network bridges purine N-3 and pyrimidine O-2 atoms in adjacent base-pairs. It is particularly regular in the A-A-T-T center, and is disrupted at the C-G-C-G ends, in part by the presence of the N-2 amino groups on guanine residues. The minor groove hydration spine may be responsible for the stability of the B form of polymers containing only A · T and I · C base-pairs, and its disruption may explain the ease of transition to the A form of polymers with G · C pairs.