Exonuclease activity is required for sequence addition and Cdc13p loading at a de novo telomere

The Saccharomyces cerevisiae Mre11p/Rad50p/Xrs2p (MRX) complex is evolutionarily conserved and functions in DNA repair and at telomeres [1-3]. In vivo, MRX is required for a 5' --> 3' exonuclease activity that mediates DNA recombination at double-strand breaks (DSBs). Paradoxically, abolition of this exonuclease activity in MRX mutants results in shortened telomeric DNA tracts. To further explore the role of MRX at telomeres, we analyzed MRX mutants in a de novo telomere addition assay in yeast cells [4]. We found that the MRX genes were absolutely required for telomerase-mediated addition in this assay. Furthermore, we found that Cdc13p, a single-stranded telomeric DNA binding protein essential for telomere DNA synthesis and protection [5], was unable to bind to the de novo telomeric DNA substrate in cells lacking Rad50p. Based on the results from this model system, we propose that the MRX complex helps to prepare telomeric DNA for the loading of Cdc13p, which then protects the chromosome from further degradation and recruits telomerase and other DNA replication components to synthesize telomeric DNA.

[1]  F. Fabre,et al.  Mutations in XRS2 and RAD50 delay but do not prevent mating-type switching in Saccharomyces cerevisiae , 1994, Molecular and cellular biology.

[2]  M. Resnick,et al.  Tying up loose ends: nonhomologous end-joining in Saccharomyces cerevisiae. , 2000, Mutation research.

[3]  J. Boeke,et al.  Designer deletion strains derived from Saccharomyces cerevisiae S288C: A useful set of strains and plasmids for PCR‐mediated gene disruption and other applications , 1998, Yeast.

[4]  T. Cech,et al.  Purification of telomerase from Euplotes aediculatus: requirement of a primer 3' overhang. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[5]  T. Lange.  Telomere Capping--One Strand Fits All , 2001 .

[6]  D. Gottschling,et al.  Telomerase-mediated telomere addition in vivo requires DNA primase and DNA polymerases alpha and delta. , 1999, Cell.

[7]  S. Evans,et al.  Est1 and Cdc13 as comediators of telomerase access. , 1999, Science.

[8]  V. Zakian,et al.  The Saccharomyces telomere-binding protein Cdc13p interacts with both the catalytic subunit of DNA polymerase alpha and the telomerase-associated est1 protein. , 2000, Genes & development.

[9]  T. Hughes,et al.  Identification of the single-strand telomeric DNA binding domain of the Saccharomyces cerevisiae Cdc13 protein. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[10]  K. Muniyappa,et al.  Telomere structure, replication and length maintenance. , 1998, Critical reviews in biochemistry and molecular biology.

[11]  T R Hughes,et al.  Cdc13p: A Single-Strand Telomeric DNA-Binding Protein with a Dual Role in Yeast Telomere Maintenance , 1996, Science.

[12]  D. Gottschling,et al.  Telomerase-Mediated Telomere Addition In Vivo Requires DNA Primase and DNA Polymerases α and δ , 1999, Cell.

[13]  James E. Haber,et al.  Telomere maintenance is dependent on activities required for end repair of double-strand breaks , 1998, Current Biology.

[14]  J. Haber,et al.  The Many Interfaces of Mre11 , 1998, Cell.

[15]  Matthias Mann,et al.  Cell-cycle-regulated association of RAD50/MRE11/NBS1 with TRF2 and human telomeres , 2000, Nature Genetics.

[16]  J. Griffith,et al.  Mammalian Telomeres End in a Large Duplex Loop , 1999, Cell.

[17]  P. Baumann,et al.  Pot1, the Putative Telomere End-Binding Protein in Fission Yeast and Humans , 2001, Science.

[18]  B. Stillman,et al.  The DNA replication fork in eukaryotic cells. , 1998, Annual review of biochemistry.

[19]  D. Gottschling,et al.  All things must end: telomere dynamics in yeast. , 2000, Cold Spring Harbor symposia on quantitative biology.

[20]  J. Petrini.  The mammalian Mre11-Rad50-nbs1 protein complex: integration of functions in the cellular DNA-damage response. , 1999, American journal of human genetics.

[21]  K. Muniyappa,et al.  Alteration of telomeric sequences and senescence caused by mutations in RAD50 of Saccharomyces cerevisiae , 1997, Genes to cells : devoted to molecular & cellular mechanisms.

[22]  J. Haber,et al.  Saccharomyces Ku70, Mre11/Rad50, and RPA Proteins Regulate Adaptation to G2/M Arrest after DNA Damage , 1998, Cell.

[23]  L. Hartwell,et al.  Single-stranded DNA arising at telomeres in cdc13 mutants may constitute a specific signal for the RAD9 checkpoint , 1995, Molecular and cellular biology.

[24]  J. Cooper.  Telomere transitions in yeast: the end of the chromosome as we know it. , 2000, Current opinion in genetics & development.

[25]  P. Philippsen,et al.  Additional modules for versatile and economical PCR‐based gene deletion and modification in Saccharomyces cerevisiae , 1998, Yeast.