Mapping the site of interaction between annexin VI and the p120GAP C2 domain

Annexin VI is a Ca(2+)-dependent membrane and phospholipid binding protein. It mediates a protein-protein interaction with the Ras p21 regulatory protein p120GAP. In this study we have mapped the binding site of GAP within the annexin VI protein. Using Far Western overlay binding assays and cell lysate competition studies we have mapped the site of interaction to the inter-lobe linker region; amino acids 325-363. Finally, using a GST fusion protein corresponding to this linker region we have demonstrated that cellular loading of the fusion protein into Rat-1 fibroblasts by electroporation blocks the interaction and co-immunoprecipitation of annexin VI and GAP.     

Yeast Rap1 contributes to genomic integrity by activating DNA damage repair genes

Rap1 (repressor-activator protein 1) is a multifunctional protein that controls telomere function, silencing and the activation of glycolytic and ribosomal protein genes. We have identified a novel function for Rap1, regulating the ribonucleotide reductase (RNR) genes that are required for DNA repair and telomere expansion. Both the C terminus and DNA-binding domain of Rap1 are required for the activation of the RNR genes, and the phenotypes of different Rap1 mutants suggest that it utilizes both regions to carry out distinct steps in the activation process. Recruitment of Rap1 to the RNR3 gene is dependent on activation of the DNA damage checkpoint and chromatin remodelling by SWI/SNF. The dependence on SWI/SNF for binding suggests that Rap1 acts after remodelling to prevent the repositioning of nucleosomes back to the repressed state. Furthermore, the recruitment of Rap1 requires TAF(II)s, suggesting a role for TFIID in stabilizing activator binding in vivo. We propose that Rap1 acts as a rheostat controlling nucleotide pools in response to shortened telomeres and DNA damage, providing a mechanism for fine-tuning the RNR genes during checkpoint activation.     

Redox state regulates binding of p53 to sequence-specific DNA, but not to non-specific or mismatched DNA.

Redox modulation of wild-type p53 plays a role in sequence-specific DNA binding in vitro . Reduction produces a DNA-binding form of the protein while oxidation produces a non-DNA-binding form. Primer extension analysis reveals that increasing concentrations of reduced p53 result in enhanced protection of the consensus sequence, while increasing concentrations of oxidized p53 confer minimal protection of the consensus sequence. DNA binding by oxidized p53 is, therefore, not sequence-specific. In contrast, there is no observable difference in the binding of oxidized p53 and reduced p53 to double-stranded non-specific or mismatched DNA in gel mobility shift assays. Both forms of p53 bind equally well, suggesting that redox modulation of p53 does not play a role in its binding to non-specific or mismatched DNA. In view of the in vitro evidence that redox state influences the sequence-specific DNA-binding of p53, we have examined the effect of oxidative stress on the in vivo ability of p53 to bind to and transactivate PG13-CAT, a reporter construct containing multiple copies of the p53 consensus binding site linked to the chloramphenicol acetyltransferase gene. Hydrogen peroxide treatment of cells cotransfected with p53 results in a marked decrease in CAT activity, suggesting that oxidation of p53 decreases the ability of the protein to bind to consensus DNA and transactivate target genes in vivo.     

Telomere Formation by Rap1p Binding Site Arrays Reveals End-Specific Length Regulation Requirements and Active Telomeric Recombination

Rap1p, the major telomere repeat binding protein in yeast, has been implicated in both de novo telomere formation and telomere length regulation. To characterize the role of Rap1p in these processes in more detail, we studied the generation of telomeres in vivo from linear DNA substrates containing defined arrays of Rap1p binding sites. Consistent with previous work, our results indicate that synthetic Rap1p binding sites within the internal half of a telomeric array are recognized as an integral part of the telomere complex in an orientation-independent manner that is largely insensitive to the precise spacing between adjacent sites. By extending the lengths of these constructs, we found that several different Rap1p site arrays could never be found at the very distal end of a telomere, even when correctly oriented. Instead, these synthetic arrays were always followed by a short ( approximately 100-bp) "cap" of genuine TG repeat sequence, indicating a remarkably strict sequence requirement for an end-specific function(s) of the telomere. Despite this fact, even misoriented Rap1p site arrays promote telomere formation when they are placed at the distal end of a telomere-healing substrate, provided that at least a single correctly oriented site is present within the array. Surprisingly, these heterogeneous arrays of Rap1p binding sites generate telomeres through a RAD52-dependent fusion resolution reaction that results in an inversion of the original array. Our results provide new insights into the nature of telomere end capping and reveal one way by which recombination can resolve a defect in this process.     

Characterization of the DNA binding features of Saccharomyces castellii Cdc13p

The principal function of Saccharomyces cerevisiae Cdc13p is to provide a loading platform to recruit complexes that provide end protection and telomere replication. We isolated the Saccharomyces castellii Cdc13p homolog (scasCdc13p) and characterized the in vitro DNA binding features of the purified recombinant scasCdc13p. The full-length scasCdc13p binds specifically to G-rich single-stranded telomeric DNA, and not to double-stranded DNA or the C-rich strand. Moreover, the minimal binding site for scasCdc13p is the octamer 5'-GTGTCTGG-3' of the S.castellii telomeric sequence. The scasCdc13p displayed a high affinity binding, where four individual nucleotide residues were found to be of most importance for the sequence specificity. Nonetheless, scasCdc13p binds the telomeric repeats from various other species, including the human. In spite of considerable divergence in telomere repeat length and sequence between these species, a conserved Cdc13p binding motif was detected. Among the budding yeasts this conserved Cdc13p binding site overlaps the Rap1p binding site. Together, these data implicate scasCdc13p as a telomere end-binding protein with a potential role in the regulation of telomere maintenance in vivo. Moreover, the results suggest that Rap1p and Cdc13p act together to preserve the conserved core present within the otherwise highly divergent btelomeric sequences among a wide variety of yeasts.     

Telomerase RNA template mutations reveal sequence-specific requirements for the activation and repression of telomerase action at telomeres

Telomeric DNA is maintained within a length range characteristic of an organism or cell type. Significant deviations outside this range are associated with altered telomere function. The yeast telomere-binding protein Rap1p negatively regulates telomere length. Telomere elongation is responsive to both the number of Rap1p molecules bound to a telomere and the Rap1p-centered DNA-protein complex at the extreme telomeric end. Previously, we showed that a specific trinucleotide substitution in the Saccharomyces cerevisiae telomerase gene (TLC1) RNA template abolished the enzymatic activity of telomerase, causing the same cell senescence and telomere shortening phenotypes as a complete tlc1 deletion. Here we analyze effects of six single- and double-base changes within these same three positions. All six mutant telomerases had in vitro enzymatic activity levels similar to the wild-type levels. The base changes predicted from the mutations all disrupted Rap1p binding in vitro to the corresponding duplex DNAs. However, they caused two classes of effects on telomere homeostasis: (i) rapid, RAD52-independent telomere lengthening and poor length regulation, whose severity correlated with the decrease in in vitro Rap1p binding affinity (this is consistent with loss of negative regulation of telomerase action at these telomeres; and (ii) telomere shortening that, depending on the template mutation, either established a new short telomere set length with normal cell growth or was progressive and led to cellular senescence. Hence, disrupting Rap1p binding at the telomeric terminus is not sufficient to deregulate telomere elongation. This provides further evidence that both positive and negative cis-acting regulators of telomerase act at telomeres.