Format

Send to

Choose Destination
Nucleic Acids Res. 2010 Apr;38(7):2444-52. doi: 10.1093/nar/gkp1206. Epub 2010 Jan 21.

The DNA binding and 3'-end preferential activity of human tyrosyl-DNA phosphodiesterase.

Author information

1
Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4255, USA.

Abstract

Human tyrosyl-DNA phosphodiesterase (Tdp1) processes 3'-blocking lesions, predominantly 3'-phosphotyrosyl bonds resulting from the trapping of topoisomerase I (Top1) cleavage complexes. The controversial ability of yeast Tdp1 to hydrolyze 5'-phosphotyrosyl linkage between topoisomerase II (Top2) and DNA raises the question whether human Tdp1 possesses 5'-end processing activity. Here we characterize the end-binding and cleavage preference of human Tdp1 using single-stranded 5'- and 3'-fluorescein-labeled oligonucleotides. We establish 3'-fluorescein as an efficient surrogate substrate for human Tdp1, provided it is attached to the DNA by a phosphodiester (but not a phosphorothioate) linkage. We demonstrate that human Tdp1 lacks the ability to hydrolyze a phosphodiester linked 5'-fluorescein. Using both fluorescence anisotropy and time-resolved fluorescence quenching techniques, we also show the preferential binding of human Tdp1 to the 3'-end. However, DNA binding competition experiments indicate that human Tdp1 binding is dependent on DNA length rather than number of DNA ends. Lastly, using surface plasmon resonance, we show that human Tdp1 selectively binds the 3'-end of DNA. Together, our results suggest human Tdp1 may act using a scanning mechanism, in which Tdp1 bind non-specifically upstream of a 3'-blocking lesion and is preferentially stabilized at 3'-DNA ends corresponding to its site of action.

PMID:
20097655
PMCID:
PMC2853120
DOI:
10.1093/nar/gkp1206
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Silverchair Information Systems Icon for PubMed Central
Loading ...
Support Center