Hus1 is one of six checkpoint Rad proteins required for all Schizosaccharomyces pombe DNA integrity checkpoints. MYC-tagged Hus1 reveals four discrete forms. The main form, Hus1-B, participates in a protein complex with Rad9 and Rad1, consistent with reports that Rad1-Hus1 immunoprecipitation is dependent on the rad9 ؉ locus. A small proportion of Hus1-B is intrinsically phosphorylated in undamaged cells and more becomes phosphorylated after irradiation. Hus1-B phosphorylation is not increased in cells blocked in early S phase with hydroxyurea unless exposure is prolonged. The Rad1-Rad9-Hus1-B complex is readily detectable, but upon cofractionation of soluble extracts, the majority of each protein is not present in this complex. Indirect immunofluorescence demonstrates that Hus1 is nuclear and that this localization depends on Rad17. We show that Rad17 defines a distinct protein complex in soluble extracts that is separate from Rad1, Rad9, and Hus1. However, two-hybrid interaction, in vitro association and in vivo overexpression experiments suggest a transient interaction between Rad1 and Rad17.

We have studied telomere length in Schizosaccharomyces pombe strains carrying mutations affecting cell cycle checkpoints, DNA repair, and regulation of the Cdc2 protein kinase. Telomere shortening was found in rad1, rad3, rad17, and rad26 mutants. Telomere lengths in previously characterized rad1 mutants paralleled the replication checkpoint proficiency of those mutants. In contrast, rad9, chk1, hus1, and cds1 mutants had intact telomeres. No difference in telomere length was seen in mutants affected in the regulation of Cdc2, whereas some of the DNA repair mutants examined had slightly longer telomeres than did the wild type. Overexpression of the rad1 ϩ gene caused telomeres to elongate slightly. The kinetics of telomere shortening was monitored by following telomere length after disruption of the rad1 ϩ gene; the rate was ϳ1 nucleotide per generation. Wild-type telomere length could be restored by reintroduction of the wildtype rad1 ϩ gene. Expression of the Saccharomyces cerevisiae RCK1 protein kinase gene, which suppresses the radiation and hydroxyurea sensitivity of Sz. pombe checkpoint mutants, was able to attenuate telomere shortening in rad1 mutant cells and to increase telomere length in a wild-type background. The functional effects of telomere shortening in rad1 mutants were assayed by measuring loss of a linear and a circular minichromosome. A minor increase in loss rate was seen with the linear minichromosome, and an even smaller difference compared with wild-type was detected with the circular plasmid.

We investigated the effects of fission yeast replication genes on telomere length maintenance and identified 20 mutant alleles that confer lengthening or shortening of telomeres. The telomere elongation was telomerase dependent in the replication mutants analyzed. Furthermore, the telomerase catalytic subunit, Trt1, and the principal initiation and lagging-strand synthesis DNA polymerase, Pol␣, were reciprocally coimmunoprecipitated, indicating these proteins physically coexist as a complex in vivo. In a pol␣ mutant that exhibited abnormal telomere lengthening and slightly reduced telomere position effect, the cellular level of the Trt1 protein was significantly lower and the coimmunoprecipitation of Trt1 and Pol␣ was severely compromised compared to those in the wild-type pol␣ cells. Interestingly, ectopic expression of wild-type pol␣ in this pol␣ mutant restored the cellular Trt1 protein to the wild-type level and shortened the telomeres to near-wild-type length. These results suggest that there is a close physical relationship between the replication and telomerase complexes. Thus, mutation of a component of the replication complex can affect the telomeric complex in maintaining both telomere length equilibrium and telomerase protein stability.The chromosome ends of almost all eukaryotes are capped by telomeres to ensure the integrity of chromosomes and to maintain the overall genome stability. Telomeres consist of simple DNA repeats, which associate with proteins to protect chromosomes from end-to-end fusion, degradation, and inappropriate recombination. Telomeric DNA consists of tandem arrays of short repeats rich in G residues on the strand that runs towards the 3Ј end of the chromosome. The telomeric tracts of all organisms are of heterogeneous length, ranging from 28 bases in some ciliates to over 50 kb in mice. However, in a given cell type, the telomeric DNA length is kept within a certain size range. A common structural feature of telomeres from different organisms is a single-stranded G-rich overhang. This G-rich overhang structure and the proteins that bind to it are thought to play an important role in mediating chromosome integrity, since in its absence, chromosome loss and fusion rates are elevated (14,32,43,49,67,72,75,77).Telomeres are also essential for the complete replication of eukaryotic chromosomes. DNA synthesis by DNA polymerases has a 5Ј-to-3Ј polarity (38). At the replication fork, the leadingstrand synthesis is thought to initiate once and then proceeds continuously. The lagging-strand synthesis is discontinuous throughout and requires repeated initiations by polymerase ␣ (Pol␣) and primase (9,12,73). Due to the polarity of DNA synthesis, the telomeric G-rich strand is synthesized by the leading-strand replication machinery, and the C-rich strand is synthesized by the lagging-strand replication machinery. Removal of the terminal RNA primer by nucleases leaves an 8-to 12-nucleotide gap at the 5Ј end of the newly replicated DNA that cannot be refilled by conventional DNA replication, r...

Serum cholinesterase 2 (CHE2) was examined in a Danish material of normal families that has been tested earlier for 70–78 classical marker systems and 25 RFLP systems. DNA for RFLP typing was provided by transforming 16‐year‐old frozen lymphocytes. The frequency of allele CHE2*C5 + in the Danish population was found to be 0.0430. The highest lod score was between CHE2 and the γ‐crystallin gene cluster (CRYG) (ẑ= 4.21 at θ= 0.00 in females). The scores were from a single family with 15 children. CHE2 may, accordingly, be assigned to the location of CRYG: chromosome 2, bands q33‐q35.