Human EZF, a Krüppel-like zinc finger protein, is expressed in vascular endothelial cells and contains transcriptional activation and repression domains.

Members of the erythroid Krüppel-like factor (EKLF) multigene family contain three C-terminal zinc fingers, and they are typically expressed in a limited number of tissues. EKLF, the founding member, transactivates the beta-globin promoter by binding to the CACCC motif. EKLF is essential for expression of the beta-globin gene as demonstrated by gene deletion experiments in mice. Using a DNA probe from the zinc finger region of EKLF, we cloned a cDNA encoding a member of this family from a human vascular endothelial cell cDNA library. Sequence analysis indicated that our clone, hEZF, is the human homologue of the recently reported mouse EZF and GKLF. hEZF is a single-copy gene that maps to chromosome 9q31. By gel mobility shift analysis, purified recombinant hEZF protein bound specifically to a probe containing the CACCC core sequence. In co-transfection experiments, we found that sense but not antisense hEZF decreased the activity of a reporter plasmid containing the CACCC sequence upstream of the thymidine kinase promoter by 6-fold. In contrast, EKLF increased the activity of the reporter plasmid by 3-fold. By fusing hEZF to the DNA-binding domain of GAL4, we mapped a repression domain in hEZF to amino acids 181-388. We also found that amino acids 91-117 of hEZF confer an activation function on the GAL4 DNA-binding domain.

Binding of Ixr1, a yeast HMG-domain protein, to cisplatin-DNA adducts in vitro and in vivo.

Ixr1 is a yeast HMG-domain protein that binds specifically to DNA adducts formed by the antitumor drug cisplatin. Interruption of the IXR1 gene in yeast desensitizes cells to cisplatin. This effect is unrelated to a natural function of Ixr1, which is to repress the transcription of COX5b. Ixr1 interacts specifically and preferentially with DNA modified by cisplatin. In the present work, Ixr1 was purified from a clone expressed in Escherichia coli. The dissociation constant for Ixr1 binding site-specifically to a 92-bp probe containing a single cis-[Pt(NH3)2{d(GpG)-N7(1) -N7(2)}] intrastrand cross-link was measured to be 2.5 (+/- 0.1) x 10(-7) M, similar to that found for HMG1. Ixr1 binds at least an order of magnitude more tightly to cisplatin-DNA adducts than to unmodified DNA. Hydroxyl radical footprinting revealed that Ixr1 protects an area of platinated DNA that is approximately 15 bp in size and centered at the platinum adduct. The binding of HMG-domain proteins to cisplatin-DNA adducts has been proposed to divert these proteins from their natural DNA-binding sites, disrupting transcription. This hypothesis was tested for Ixr1 in yeast. The protein was not titrated away from the Cox5b promoter sufficiently well to disrupt transcription either of Cox5b mRNA from genomic DNA or of the beta-galactosidase gene under control of the promoter in a plasmid DNA transformed into yeast.

The HMG-domain protein Ixr1 blocks excision repair of cisplatin-DNA adducts in yeast.

Ixr1 is a yeast HMG-domain protein which binds the major DNA adducts of the antitumor drug cisplatin. Previous work demonstrated that Saccharomyces cerevisiae cells lacking the IXR1 gene were two-fold less sensitive to cisplatin treatment than wild-type cells, and the present investigation reveals a six-fold difference in yeast having a different background. The possibility that the lower cytotoxicity of cisplatin in the ixr1 strain is the result of enhanced repair was investigated in rad1, rad2, rad4, rad6, rad9, rad10, rad14 and rad52 backgrounds. In three of the excision repair mutants, rad2, rad4 and rad14, the differential sensitivity caused by removing the Ixr1 protein was nearly abolished. This result demonstrates that the greater cisplatin resistance in the ixr1 strain is most likely a consequence of excision repair, supporting the theory that Ixr1 and other HMG-domain proteins can block repair of the major cisplatin-DNA adducts in vivo. The differential sensitivity of wild-type cells and those lacking Ixr1 persisted in the rad1 and rad10 strains, however, indicating that these two proteins act at a stage in the excision repair pathway where damage recognition is less critical. A model is proposed to account for these results, which is strongly supported recently identified functional roles for the rad excision repair gene products. A rad52 mutant was more sensitive to cisplatin than the RAD52 parental strain, which reveals that Rad52, a double-strand break repair protein, repairs cisplatin-DNA adducts, probably interstrand cross-links. A rad52 ixr1 strain was less sensitive to cisplatin than the rad52 IXR1 strain, consistent with Ixr1 not blocking repair of cisplatin adducts removed by Rad52 rad6 strains behaved similarly, except they were both substantially more sensitive to cisplatin. Interruption of the RAD9 gene, which is involved in DNA-damage-induced cell cycle arrest, had no affect on cisplatin cytotoxicity.