Phosphorylation mutants of p53 show differential complex formation with putative dehydrogenase Tms1 of fission yeast

The yeast tms1 gene was originally identified as a multi-copy suppressor of a lethal growth arrest caused by expression of a tumour mutant cDNA of p53 in fission yeast. The tms1 gene product (Tms1) was found to form stable complexes with p53 in yeast and in vitro; using purified recombinant proteins, the interaction was mapped to the C-terminal region of p53. This part is known to be modified by several protein kinases resulting in a transition of p53 from a latent to an activated state capable of transactivating various cellular genes involved in growth suppression or apoptosis. Since there is evidence for an evolutionary conservation of a Tms1-related protein in mammals, the effect of the phosphorylation status of the C-terminus of p53 on Tms1/p53 complex formation in vitro has been investigated. Whereas mutants changing the cdc2 phosphorylation site at position 315 of human p53 had only little effect on Tms1/p53 complex formation, we found that mutants involving the protein kinase CK2 site at position 392 showed a significantly decreased relative affinity for the Tms1 protein. The same result was obtained by using a C-terminal fragment of p53 which was phosphorylated by purified protein kinase CK2, suggesting that the complex formation of p53 with cellular C-terminal binding proteins like Tms1 impairs regulation by phosphorylation.     

Human CTLA-4 is expressed in situ on T lymphocytes in germinal centers, in cutaneous graft-versus-host disease, and in Hodgkin''s disease

We have previously reported that the incision efficiency of the nucleotide excision repair (NER) reaction measured in vitro with cell-free human protein extracts was reduced by up to 80% on a linearized damaged plasmid DNA substrate when compared to supercoiled damaged DNA. The inhibition stemed from the presence of the DNA-end binding Ku70/Ku80 heterodimer which is the regulatory subunit of the DNA-dependent protein kinase (DNA-PK). Here, the origin of the repair inhibition was assessed by a new in vitro assay in which circular or linear plasmid DNA, damaged or undamaged, was quantitatively adsorbed on sensitized microplate wells. The binding of two NER proteins, XPA and p62-TFIIH, indispensable for the incision step of the reaction, was quantified either directly in an ELISA-like reaction in the wells with specific antibodies or in Western blotting experiments on the DNA-bound fraction. We report a dramatic inhibition of XPA and p62-TFIIH association with UVC photoproducts on linear DNA. XPA and p62-TFIIH binding to DNA damage was regained when the reaction was performed with extracts lacking Ku activity (extracts from xrs6 rodent cells) whereas addition of purified human Ku complex to these extracts restored the inhibition. Despite the fact that DNA-PK was active during the NER reaction, the mechanism of inhibition relied on the sole Ku complex, since mutant protein extracts lacking the catalytic DNA-PK subunit (extracts from the human M059J glioma cells) exhibited a strong binding inhibition of XPA and p62-TFIIH proteins on linear damaged DNA, identical to the inhibition observed with the DNA-PK+ control extracts (from M059K cells).     

Flagellar motor-switch binding face of CheY and the biochemical basis of suppression by CheY mutants that compensate for motor-switch defects in Escherichia coli

CheY is a response regulator protein of Escherichia coli that interacts with the flagellar motor-switch complex to modulate flagellar rotation during chemotaxis. The switch complex is composed of three proteins, FliG, FliM, and FliN. Recent biochemical data suggest a direct interaction of CheY with FliM. In order to determine the FliM binding face of CheY, we isolated dominant suppressors of fliM mutations in cheY with limited allele specificity. The protein products of suppressor cheY alleles were purified and assayed for FliM binding. Six out of nine CheY mutants were defective in FliM binding. Suppressor amino acid substitutions were mapped on the crystal structure of CheY showing clustering of reduced binding mutations on a solvent-accessible face of CheY, thus revealing a FliM binding face of CheY. To examine the basis of genetic suppression, we cloned, purified, and tested FliM mutants for CheY binding. Like the wild-type FliM, the mutants were also defective in binding to various CheY suppressor mutants. This was not expected if CheY suppressors were compensatory conformational suppressors. Furthermore, a comparison of flagellar rotation patterns indicated that the cheY suppressors had readjusted the clockwise bias of the fliM strains. However, a chemotaxis assay revealed that the readjustment of the clockwise bias was not sufficient to make cells chemotactic. Although the suppressors did not restore chemotaxis, they did increase swarming on motility plates by a process called "pseudotaxis." Therefore, our genetic selection scheme generated suppressors of pseudotaxis or switch bias adjustment. The binding results suggest that the mechanism for this adjustment is the reduction in binding affinity of activated CheY. Therefore, these suppressors identified the switch-binding surface of CheY by loss-of-function defects rather than gain-of-function compensatory conformational changes.     

DNA-binding activities of Hop1 protein, a synaptonemal complex component from Saccharomyces cerevisiae

The meiosis-specific HOP1 gene is important both for crossing over between homologs and for production of viable spores. hop1 diploids fail to assemble synaptonemal complex (SC), which normally provides the framework for meiotic synapsis. Immunochemical methods have shown that the 70-kDa HOP1 product is a component of the SC. To assess its molecular function, we have purified Hop1 protein to homogeneity and shown that it forms dimers and higher oligomers in solution. Consistent with the zinc-finger motif in its sequence, the purified protein contained about 1 mol equivalent of zinc whereas mutant protein lacking a conserved cysteine within this motif did not. Electrophoretic gel mobility shift assays with different forms of M13 DNA showed that Hop1 binds more readily to linear duplex DNA and negatively superhelical DNA than to nicked circular duplex DNA and even more weakly to single-stranded DNA. Linear duplex DNA binding was enhanced by the addition of Zn2+, was stronger for longer DNA fragments, and was saturable to about 55 bp/protein monomer. Competitive inhibition of this binding by added oligonucleotides suggests preferential affinity for G-rich sequences and weaker binding to poly(dA-dT). Nuclear extracts of meiotic cells caused exonucleolytic degradation of linear duplex DNA if the extracts were prepared from hop1 mutants; addition of purified Hop1 conferred protection against this degradation. These findings suggest that Hop1 acts in meiotic synapsis by binding to sites of double-strand break formation and helping to mediate their processing in the pathway to meiotic recombination.