Characterization of the S. cerevisiae Rap1 DNA binding domain and biochemical analysis of its interactions with telomeric and silencer DNA

ABSTRACT The Sacharomyces cerevisiae gene product Rapl is a multifunctional protein that binds D N A via a 235 amino acid domain. Rapl binds in vivo to telomeres and silent mating type genes, where the protein anchors a heterochromatin complex. Two Helix-Turn-Helix (HTH) motifs within the DNA-binding domain (DBD) have been elucidated by X-ray crystallography (Konig etal., 1996). In work performed before a crystal structure of D B D was available, weak identity between portions of the DBD and the c-Myb D N A binding motif was used to align the two sequences. Conservation of key hydrophobic residues comprising the c-Myb H T H "core" was used to confirm functional identity. The second H T H motif (HTH2), located C-terminal to HTH1, was also studied. Analysis of the DBD revealed a highly conserved stretch of amino acids with functional identity to the HTH1 recognition helix. This HTH2 recognition helix was then modeled from various data. After the DBD crystal structure determination (Konig et al., 1996), a second set of experiments was performed to study the affinity of Rapl for telomere and silencer sites. Dissociation constant (Kd) data showed high affinity (1X10" M) of Rapl for telomere and silencer H M L E binding sites in vitro. Competition assays to generate apparent IC 5 0 values provided qualitative data on the relative affinity of Rapl for telomere, H M R E silencer and H M L E silencer sites. Rapl showed slightly reduced affinity for H M L E sites. In vivo studies of Rapl binding were performed using the CAT mammalian assay system as a reporter. These studies showed that Rapl binds with higher affinity to telomeric than to silencer sites. A variety of RAP1 site-directed mutants were also assayed for Kd, IC 5 0 and CAT activity. Proteins bearing mutations affecting HTH1 were found to bind telomeres with high affinity, but a dramatic loss in binding was seen with H M L E sites. These data, combined with experiments using altered telomere and HMR-E sites, suggested that HTH1 and HTH2 bind D N A in a cooperative manner: the affinity of one subdomain is enhanced when the second subdomain is bound to DNA. Expression of HTH 1 in E. coli resulted in a protein able to bind D N A only non-specifically. Deletion of a "tail" region at the N-terminus of the DBD was also performed. This region has been implicated in D N A binding and makes base-specific contacts in the majorThe Sacharomyces cerevisiae gene product Rapl is a multifunctional protein that binds D N A via a 235 amino acid domain. Rapl binds in vivo to telomeres and silent mating type genes, where the protein anchors a heterochromatin complex. Two Helix-Turn-Helix (HTH) motifs within the DNA-binding domain (DBD) have been elucidated by X-ray crystallography (Konig etal., 1996). In work performed before a crystal structure of D B D was available, weak identity between portions of the DBD and the c-Myb D N A binding motif was used to align the two sequences. Conservation of key hydrophobic residues comprising the c-Myb H T H "core" was used to confirm functional identity. The second H T H motif (HTH2), located C-terminal to HTH1, was also studied. Analysis of the DBD revealed a highly conserved stretch of amino acids with functional identity to the HTH1 recognition helix. This HTH2 recognition helix was then modeled from various data. After the DBD crystal structure determination (Konig et al., 1996), a second set of experiments was performed to study the affinity of Rapl for telomere and silencer sites. Dissociation constant (Kd) data showed high affinity (1X10" M) of Rapl for telomere and silencer H M L E binding sites in vitro. Competition assays to generate apparent IC 5 0 values provided qualitative data on the relative affinity of Rapl for telomere, H M R E silencer and H M L E silencer sites. Rapl showed slightly reduced affinity for H M L E sites. In vivo studies of Rapl binding were performed using the CAT mammalian assay system as a reporter. These studies showed that Rapl binds with higher affinity to telomeric than to silencer sites. A variety of RAP1 site-directed mutants were also assayed for Kd, IC 5 0 and CAT activity. Proteins bearing mutations affecting HTH1 were found to bind telomeres with high affinity, but a dramatic loss in binding was seen with H M L E sites. These data, combined with experiments using altered telomere and HMR-E sites, suggested that HTH1 and HTH2 bind D N A in a cooperative manner: the affinity of one subdomain is enhanced when the second subdomain is bound to DNA. Expression of HTH 1 in E. coli resulted in a protein able to bind D N A only non-specifically. Deletion of a "tail" region at the N-terminus of the DBD was also performed. This region has been implicated in D N A binding and makes base-specific contacts in the major