Wapl is a new regulator of sister chromatid resolution and promotes release of cohesin from chromosomes by directly interacting with its regulatory subunits.

Human telomeres bind shelterin, the six-subunit protein complex that protects chromosome ends from the DNA damage response and regulates telomere length maintenance by telomerase. We used quantitative immunoblotting to determine the abundance and stoichiometry of the shelterin proteins in the chromatin-bound protein fraction of human cells. The abundance of shelterin components was similar in primary and transformed cells and was not correlated with telomere length. The duplex telomeric DNA binding factors in shelterin, TRF1 and TRF2, were sufficiently abundant to cover all telomeric DNA in cells with short telomeres. The TPP1⅐POT1 heterodimer was present 50 -100 copies/telomere, which is in excess of its singlestranded telomeric DNA binding sites, indicating that some of the TPP1⅐POT1 in shelterin is not associated with the singlestranded telomeric DNA. TRF2 and Rap1 were present at 1:1 stoichiometry as were TPP1 and POT1. The abundance of TIN2 was sufficient to allow each TRF1 and TRF2 to bind to TIN2. Remarkably, TPP1 and POT1 were ϳ10-fold less abundant than their TIN2 partner in shelterin, raising the question of what limits the accumulation of TPP1⅐POT1 at telomeres. Finally, we report that a 10-fold reduction in TRF2 affects the regulation of telomere length but not the protection of telomeres in tumor cell lines.Telomeres solve the two main problems associated with the organization of eukaryotic genetic information on linear chromosomes: that is, the end-replication problem and the endprotection problem. The end-replication problem is solved by the interaction of telomeres with telomerase, a telomerespecific reverse transcriptase that can replenish telomeric sequences lost during DNA replication (1). Mammalian telomeres solve the end-protection problem through their association with shelterin, a six-protein complex that specifically associates with telomeric DNA and represses both DNA damage signaling and double-strand break repair reactions at the chromosome ends (2).Shelterin binds to the duplex telomeric TTAGGG repeat array through two related DNA-binding proteins, TRF1 and TRF2 (3-5). A third DNA binding factor in shelterin, POT1, binds to single-stranded TTAGGG repeats (6). A 50 -400-nucleotide stretch of single-stranded DNA is found at the 3Ј terminus of telomeres (7) or at a telomere-internal displacement loop (D loop) that is formed by strand-invasion of the 3Ј overhang when telomeres are in the t-loop configuration (8). In vitro, POT1 can bind to TTAGGG repeats both at a 3Ј end and at the non-terminal sites found in the D loop (9 -12). The POT1 DNA binding activity is enhanced by its binding partner TPP1 (9 -12). TPP1 also connects POT1 to TIN2, which binds to TRF1 and TRF2 (13-19). The sixth component of human shelterin, Rap1, interacts with TRF2 (20).The view that the shelterin components can form a single six-subunit complex at telomeres has emerged from both the biochemical purification of the six-protein complex and studies of the protein-protein interaction network, including the finding t...

Kinesin-I is essential for the transport of membrane-bound organelles in neural and nonneural cells. However, the means by which kinesin interacts with its intracellular cargoes, and the means by which kinesin-cargo interactions are regulated in response to cellular transport requirements are not fully understood. The C terminus of the Drosophila kinesin heavy chain (KHC) was used in a two-hybrid screen of a Drosophila cDNA library to identify proteins that bind specifically to the kinesin tail domain. UNC-76 is an evolutionarily conserved cytosolic protein that binds to the tail domain of KHC in two-hybrid and copurification assays, indicating that kinesin and UNC-76 form a stable complex in vivo. Loss of Drosophila Unc-76 function results in locomotion and axonal transport defects reminiscent of the phenotypes observed in kinesin mutants, suggesting that UNC-76 is required for kinesin-dependent axonal transport. Unc-76 exhibits dosage-sensitive genetic relationships with Khc and Kinesin light chain mutations, further supporting the hypothesis that UNC-76 and kinesin-I work in a common transport pathway. Given the interaction of FEZ1, the mammalian homolog of UNC-76, with protein kinase Czeta, and the role of FEZ1 in axon outgrowth, we propose that UNC-76 helps integrate kinesin activity in response to transport requirements in axons.

We have performed a mutational analysis together with RNA interference to determine the role of the kinesin-like protein KLP67A in Drosophila cell division. During both mitosis and male meiosis, Klp67A mutations cause an increase in MT length and disrupt discrete aspects of spindle assembly, as well as cytokinesis. Mutant cells exhibit greatly enlarged metaphase spindle as a result of excessive MT polymerization. The analysis of both living and fixed cells also shows perturbations in centrosome separation, chromosome segregation, and central spindle assembly. These data demonstrate that the MT plus end-directed motor KLP67A is essential for spindle assembly during mitosis and male meiosis and suggest that the regulation of MT plus-end polymerization is a key determinant of spindle architecture throughout cell division.

No abstract