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DNA double strand break (DSB) repair by non-homologous end joining (NHEJ) in mammalian cells requires the Ku70-Ku80 heterodimer, the DNA-PK catalytic subunit DNA-PKcs, as well as DNA ligase IV and Xrcc4. NHEJ of plasmid DSBs in Saccharomyces cerevisiae requires Ku, Xrcc4 and DNA ligase IV, as well as Mre11, Rad50, Xrs2 and DNA damage checkpoint proteins. Saccharomyces cerevisiae Ku is also required for telomere length maintenance and transcriptional silencing. We have characterized NHEJ in Schizosaccharomyces pombe using an extrachromosomal assay and find that, as anticipated, it is Ku70 and DNA ligase IV dependent. Unexpectedly, we find that Rad32, Rad50 (the S.pombe homologues of Mre11 and Rad50, respectively) and checkpoint proteins are not required for NHEJ. Furthermore, although S.pombe Ku70 is required for maintenance of telomere length, it is dispensable for transcriptional silencing at telomeres and is located throughout the nucleus rather than concentrated at the telomeres. Together, these results provide insight into the mechanism of NHEJ and contrast significantly with recent studies in S.cerevisiae.

The sequence requirements for in vivo telomere function in the fission yeast, Schizosaccharomyces pombe, have been investigated. A 258 bp tract of previously characterized cloned fission yeast terminal repeats adjacent to 800 bp of telomere-associated sequences is sufficient to seed new telomeres onto linearized ars-containing plasmids when introduced into cells. The resulting transformants contain unrearranged, acentric, linear episomes. Cloned telomeres, with and without telomere-associated sequences adjacent to the 258 bp terminal repeats, were utilized to introduce chromosome breaks at specific sites in a non-essential minichromosome. Truncated minichromosome derivatives were recovered containing the ura4 or ade6 gene adjacent to a newly formed telomere. These telomeres exert reversible position effects on the expression of the adjacent ura4 or ade6 genes.