Characterization of the Escherichia coli damage-independent UvrBC endonuclease activity.

Incision of damaged DNA templates by UvrBC in Escherichia coli depends on UvrA, which loads UvrB on the site of the damage. A 50-base pair 3' prenicked DNA substrate containing a cholesterol lesion is incised by UvrABC at two positions 5' to the lesion, the first incision at the eighth and the second at the 15th phosphodiester bond. Analysis of a 5' prenicked cholesterol substrate revealed that the second 5' incision is efficiently produced by UvrBC independent of UvrA. This UvrBC incision was also found on the same substrate without a lesion and, with an even higher efficiency, on a DNA substrate containing a 5' single strand overhang. Incision occurred in the presence of ATP or ADP but not in the absence of cofactor. We could show an interaction between UvrB and UvrC in solution and subsequent binding of this complex to the substrate with a 5' single strand overhang. Analysis of mutant UvrB and UvrC proteins revealed that the damage-independent nuclease activity requires the protein-protein interaction domains, which are exclusively needed for the 3' incision on damaged substrates. However, the UvrBC incision uses the catalytic site in UvrC which makes the 5' incision on damaged DNA substrates.

Isolation and identification of the human homolog of a new p53-binding protein, Mdmx.

We recently reported the identification of a mouse cDNA encoding a new p53-associating protein that we called Mdmx because of its structural similarity to Mdm2, a well-known p53-binding protein. Here we report the isolation of a cDNA encoding the human homolog of Mdmx. The ORF of the cDNA encodes a protein of 490 amino acids, 90% similar to mouse Mdmx. The homology between Mdmx and Mdm2 is most prominent in the p53-binding domain and the putative metal-binding domains. The Mdmx protein, which, based on SDS-PAGE, has a MW of 80 kDa, can bind p53 in vitro. The human MDMX gene is transcribed in all tissues tested, with high levels in thymus. By fluorescence in situ hybridization analysis we mapped the mouse mdmx gene to chromosome 1 (region F-G) and the human MDMX gene to chromosome 1q32.

MDMX: a novel p53-binding protein with some functional properties of MDM2.

Here we report the isolation of a cDNA encoding a new p53-associating protein. This new protein has been called MDMX on the basis of its structural similarity to MDM2, which is especially notable in the p53-binding domain. In addition, the putative metal binding domains in the C-terminal part of MDM2 are completely conserved in MDMX. The middle part of the MDMX and MDM2 proteins shows a low degree of conservation. We can show by co-immunoprecipitation that the MDMX protein interacts specifically with p53 in vivo. This interaction probably occurs with the N-terminal part of p53, because the activity of the transcription activation domain of p53 was inhibited by co-transfection of MDMX. Northern blotting showed that MDMX, like MDM2, is expressed in all tissues tested, and that several mRNAs for MDMX can be detected. Interestingly, the level of MDMX mRNA is unchanged after UV irradiation, in contrast to MDM2 transcription. This observation suggests that MDMX may be a differently regulated modifier of p53 activity in comparison with MDM2. Our study indicates that at least one additional member of the MDM protein family exists which can modulate p53 function.