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Sucl+ was originally identified as a DNA sequence that, at high copy number, rescued Schizosaccharomyces pombe strains carrying certain temperature-sensitive alleles of the cdc2 cell cycle control gene. We determined the nucleotide sequence of a 1,083-base-pair Sucl+ DNA fragment and Si mapped its 866-nucleotide RNA transcript. The protein-coding sequence of the gene is interrupted by two intervening sequences of 115 and 51 base pairs. The predicted translational product of the gene is a protein of 13 kilodaltons. A chromosomal gene disruption of Sucl+ was constructed in a diploid S. pombe strain. Germinating spores carrying a null allele of the gene were capable of very limited cell division, following which many cells became highly elongated. The Sucl+ gene was also strongly overexpressed under the control of a heterologous S. pombe promoter. Overexpression of Sucl+ is not lethal but causes a division delay such that cells are approximately twice the normal length at division. These data suggest that Sucl+ encodes a protein which plays a direct role in the cell division cycle of S. pombe.Among the many genes required for completion of the cell division cycle in the fission yeast Schizosaccharomyces pombe, the cdc2+ gene is of particular interest because it plays a role in both the G, and the G2 phases of the cell cycle. The activity of the gene is required in G, before DNA replication and in G2 before initiation of mitosis (24). There is also physiological and genetic evidence that cdc2+ is not only required in G, and G2 but acts at the rate-limiting step which controls the rate of progression into the S phase and nuclear division (25).The gene product of cdc2+ shares 62% sequence homology with the product of the CDC28 cell cycle "start" gene of the distantly related budding yeast Saccharomyces cerevisiae (15,18). Furthermore, the CDC28 gene can rescue cdc2(Ts) mutants of S. pombe (1), and the cdc2+ gene can rescue cdc28(Ts) strains of S. cerevisiae (7). Both genes encode products which have protein kinase activity (26, 30).The cdc2+ gene was physically isolated from a gene bank of S. pombe DNA by transformation and rescue of a temperature-sensitive cdc2-33 S. pombe strain (1). During screening for DNA sequences which restored a cdc+ phenotype to a cdc2-33 strain, not only cdc2+ but also a second, previously unidentified gene known as Sucl+ was isolated (14). The Sucl + gene, carried on a multicopy number vector pDB248 (2), allowed a cdc2-33 strain to propagate at a nonpermissive temperature. This effect was found to be specific for certain alleles of cdc2. Of the temperaturesensitive alleles tested, cdc2-33, cdc2-56, and cdc2-L7 were rescued by Sucl , whereas cdc2-M35, cdc2-M63, cdc2-M26, and cdc2-M55 were not rescued (14).It has been suggested that the Sucl+ gene product interacts with the product of cdc2+, but there is presently no evidence that Sucl+ plays a direct role in the cell division cycle. In the present study, we determined the nucleotide sequence of Sucl+, identified its protein coding sequence, ...

We have isolated and characterised the pht1 gene from the fission yeast Schizosaccharomyces pombe. The sequence of the predicted translation product has revealed a striking similarity to the family of H2A.F/Z histone variant proteins, which have been found in a variety of different organisms. Cells deleted for the pht1 gene locus grow slowly, exhibit an altered colony morphology, increased resistance to heat shock and show a significant decrease in the fidelity of segregation of an S. pombe minichromosome. We propose that the histone H2A variant encoded by the pht1 gene is important for chromosomal structure and function, possibly including a role in controlling the fidelity of chromosomal segregation during mitosis.

To examine the factors governing the generation of DNA sequence rearrangements in mammalian somatic cells, we have cloned and sequenced novel junctions produced by six spontaneous deletion mutations at the aprt locus of Chinese hamster ovary cells. Our analyses indicate that these rearrangements were produced by non‐homologous recombinational events occurring between short (2‐7 bp) sequence repeats at the two termini of the deletion which leave one copy of the repeat in the mutant gene. Certain tri‐ and tetranucleotides recur at the deletion termini, suggesting that these may possibly be a recognition sequence for an enzyme involved in the event. No other gene structural alterations were found at the novel junctions or in neighbouring sequences. The deletions are not randomly distributed over the aprt gene; four termini clustered in a 40‐bp sequence. This region of aprt is unusual as it contains both significant stretches of dyad symmetry which could potentially form stable DNA secondary structures and short direct repeats. Regions of dyad symmetry were also found at at least one terminus of all the deletions. In view of the similar properties of this set of deletions, possible mechanisms for the formation of this type of gene rearrangement are considered.

The interaction of ataxia-telangiectasia mutated (ATM) and the Mre11/Rad50/Nbs1 (MRN) complex is critical for the response of cells to DNA double-strand breaks; however, little is known of the role of these proteins in response to DNA replication stress. Here, we report a mutant allele of MRE11 found in a colon cancer cell line that sensitizes cells to agents causing replication fork stress. The mutant Mre11 weakly interacts with Rad50 relative to wild type and shows little affinity for Nbs1. The mutant protein lacks 3-5 exonuclease activity as a result of loss of part of the conserved nuclease domain; however, it retains binding affinity for single-stranded DNA (ssDNA), double-stranded DNA with a 3 singlestrand overhang, and fork-like structures containing ssDNA regions. In cells, the mutant protein shows a time-and dose-dependent accumulation in chromatin after thymidine treatment that corresponds with increased recruitment and hyperphosphorylation of replication protein A. ATM autophosphorylation, Mre11 foci, and thymidine-induced homologous recombination are suppressed in cells expressing the mutant allele. Together, our results suggest that the mutant Mre11 suppresses the cellular response to replication stress by binding to ssDNA regions at disrupted forks and impeding replication restart in a dominant negative manner. INTRODUCTIONThe MRN complex, consisting of Mre11, Rad50 and NBS1, has diverse functions in DNA damage recognition (Petrini and Stracker, 2003), DNA replication (Costanzo et al., 2001), cell cycle checkpoint activation (Grenon et al., 2001), nonhomolgous end joining (Paull and Gellert, 2000), and telomere maintenance (Wu et al., 2007). The Mre11 complex binds DNA double-strand breaks (DSBs) soon after they are formed implicating it in DNA damage detection . Furthermore, the complex can tether linear duplex molecules (de Jager et al., 2001), and it is able to bridge broken DNA ends or sister chromatids (van den Bosch et al., 2003). Mre11 has 3Ј-5Ј exonuclease activity and endonuclease activity (Paull and Gellert, 1999), suggesting a role in the processing of DNA ends into forms that can be recognized by cell cycle checkpoint and DNA repair proteins (Paull and Gellert, 1999;Lee and Paull, 2005;Jazayeri et al., 2006). However, precise cellular roles of the Mre11 complex have been difficult to establish, because null mutations of all components of the complex are lethal to vertebrate cells (Luo et al., 1999;Yamaguchi-Iwai et al., 1999;Zhu et al., 2001).There are several lines of evidence implicating the MRN complex in DNA replication. The complex associates with chromatin and colocalizes with proliferating cell nuclear antigen (PCNA) throughout S phase (Maser et al., 2001). In addition, chromatin loading of Mre11 is enhanced by fork stalling, suggesting that the complex is loaded at the replication fork (Mirzoeva and Petrini, 2003). Depletion of Mre11 from DT40 or Xenopus leads to increased chromosomal breaks and accumulation of DSBs during DNA replication (Yamaguchi-Iwai et al., 1999;Costanzo et...