Characterization of a novel human SMC heterodimer homologous to the Schizosaccharomyces pombe Rad18/Spr18 complex.

The structural maintenance of chromosomes (SMC) protein encoded by the fission yeast rad18 gene is involved in several DNA repair processes and has an essential function in DNA replication and mitotic control. It has a heterodimeric partner SMC protein, Spr18, with which it forms the core of a multiprotein complex. We have now isolated the human orthologues of rad18 and spr18 and designated them hSMC6 and hSMC5. Both proteins are about 1100 amino acids in length and are 27-28% identical to their fission yeast orthologues, with much greater identity within their N- and C-terminal globular domains. The hSMC6 and hSMC5 proteins interact to form a tight complex analogous to the yeast Rad18/Spr18 heterodimer. In proliferating human cells the proteins are bound to both chromatin and the nucleoskeleton. In addition, we have detected a phosphorylated form of hSMC6 that localizes to interchromatin granule clusters. Both the total level of hSMC6 and its phosphorylated form remain constant through the cell cycle. Both hSMC5 and hSMC6 proteins are expressed at extremely high levels in the testis and associate with the sex chromosomes in the late stages of meiotic prophase, suggesting a possible role for these proteins in meiosis.

Core-directed protein design. I. An experimental method for selecting stable proteins from combinatorial libraries.

The design of proteins represents a significant challenge to modern-day structural biology. A major obstacle here is the specification of well-packed hydrophobic cores to drive the folding and stabilization of the target. Computational approaches have been used to alleviate this by testing alternate sequences prior to the production and characterization of a few proteins. Here we present the experimental counterpart of this approach. We selected stable variants from a library of ubiquitin hydrophobic-core mutants as follows. Hexahistidine-tagged proteins were displayed on the surface of phage. These protein-phage were immobilized onto Ni-coated surfaces. The bound fusion-phage were treated with protease to remove unstable or poorly folded proteins. Stable phage fusions were eluted and infected into Escherichia coli, which allowed amplification for further selection, sequencing, or protein expression. Two Ni-derivatized supports were tested: Ni-NTA chips for surface plasmon resonance (SPR) and Ni-NTA agarose beads. SPR had an advantage in that the selection process could be monitored directly. This allowed individual clones and experimental conditions to be tested rapidly prior to preparative panning of the library, which was carried out using Ni-NTA agarose beads. We demonstrate the method by selecting stable core mutants of ubiquitin, the characterization of which is described in the following paper [Finucane, M. D., and Woolfson, D. N. (1999) Biochemistry 38, XXXXX-XXXXX]. As our method selects only on the basis of structure and stability, it will be of use in improving the stabilities and structural specificities of proteins of de novo design, and in establishing rules that link sequence and structure.

Double-strand cleavage at a two-base deletion mismatch in a DNA heteroduplex by nuclease S1.

A two-base deletion mismatch was generated in a DNA heteroduplex by hybridization of two linear plasmid DNA molecules differing only by the presence of a two-base deletion in one of them. The heteroduplex was shown to be sensitive to double-strand cleavage by nuclease S1, thus demonstrating the potential value of single-stranded probes for the detection of polymorphisms in genomic DNA due to very small deletions.