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Cisplatin-DNA adducts are molecular decoys for the ribosomal RNA transcription factor hUBF (human upstream binding
D K Treiber1, X Zhai, H M Jantzen
1Department of Chemistry, Massachusetts Institute of Technology, Cambridge 02139.
Abstract:
The toxicity of DNA-damaging agents is widely believed to result from the formation of lesions that block polymerases or disrupt the integrity of the genome. A mechanism heretofore not addressed is that DNA damage may titrate essential DNA-binding proteins away from their natural sites of action. This report shows that the ribosomal RNA (rRNA) transcription factor hUBF (human upstream binding factor) binds with striking affinity (Kd(app) approximately 60 pM) to the intrastrand cis-[Pt(NH3)2](2+-d(GpG) crosslink formed by the anticancer drug cis-diamminedichloroplatinum(II) (cisplatin). When protein blots of human cell extracts are probed with cisplatin-modified DNA, 97- and 94-kDa proteins are detected, consistent with the known sites of hUBF species. A similar analysis of blots containing in vitro translated hUBF confirmed that the protein binds cisplatin adducts with high specificity. By contrast, DNA adducts of the clinically ineffective trans isomer of cisplatin, trans-diamminedichloroplatinum(II), are not recognized by hUBF. DNase I inhibition patterns of hUBF bound to a 100-base-pair DNA fragment containing a centrally located cis-[Pt(NH3)2](2+)-d(GpG) crosslink reveal specific protein-DNA interactions in a 14-base-pair region flanking the adduct. The affinity of hUBF for the rRNA promoter is similar (Kd(app) approximately 18 pM) to that measured for the cisplatin adduct. In addition, we observe that the hUBF-promoter interaction is highly sensitive to the antagonistic effects of cisplatin-DNA adducts. These results suggest that a cisplatin-mediated transcription-factor-hijacking mechanisms could disrupt rRNA synthesis, which is stimulated in proliferating cells.
Insights
Anticancer drug cisplatin binds strongly to DNA, sequestering the ribosomal RNA transcription factor hUBF. This DNA damage mechanism may disrupt essential rRNA synthesis in proliferating cells.
Area of Science:
- Molecular Biology
- Cancer Research
- Biochemistry
Background:
- DNA-damaging agents are thought to cause toxicity by forming lesions that block polymerases or disrupt genome integrity.
- A less-explored mechanism is the titration of essential DNA-binding proteins away from their natural sites by DNA damage.
Purpose of the Study:
- To investigate the interaction between the ribosomal RNA (rRNA) transcription factor hUBF (human upstream binding factor) and DNA adducts formed by the anticancer drug cisplatin.
- To determine if cisplatin-DNA adducts can sequester hUBF and potentially disrupt rRNA synthesis.
Main Methods:
- Probing protein blots of human cell extracts and in vitro translated hUBF with cisplatin-modified DNA to detect protein binding.
- Using DNase I inhibition assays to map the interaction sites between hUBF and DNA containing cisplatin adducts.
- Comparing the binding affinity of hUBF to cisplatin adducts and the rRNA promoter.
Main Results:
- Human upstream binding factor (hUBF) exhibits high affinity for the intrastrand cis-[Pt(NH3)2](2+)-d(GpG) crosslink formed by cisplatin (Kd(app) approx. 60 pM).
- Specific hUBF species (97 and 94 kDa) were detected binding to cisplatin-modified DNA.
- hUBF binding to cisplatin adducts was highly specific, with no recognition of adducts formed by the trans isomer.
- hUBF binding to the rRNA promoter was sensitive to the presence of cisplatin-DNA adducts.
Conclusions:
- Cisplatin-DNA adducts can effectively sequester the rRNA transcription factor hUBF.
- This sequestration represents a novel mechanism of cisplatin toxicity, potentially disrupting rRNA synthesis.
- The disruption of rRNA synthesis may be particularly relevant in rapidly proliferating cells, which rely heavily on rRNA production.