DNase I footprinting and enhanced exonuclease function of the bipartite Werner syndrome protein (WRN) bound to partially melted duplex DNA.

Abstract

Werner syndrome is a premature aging and cancer-prone hereditary disorder caused by deficiency of the WRN protein that harbors 3' -->5' exonuclease and RecQ-type 3' --> 5' helicase activities. To assess the possibility that WRN acts on partially melted DNA intermediates, we constructed a substrate containing a 21-nucleotide noncomplementary region asymmetrically positioned within a duplex DNA fragment. Purified WRN shows an extremely efficient exonuclease activity directed at both blunt ends of this substrate, whereas no activity is observed on a fully duplex substrate. High affinity binding of full-length WRN protects an area surrounding the melted region of the substrate from DNase I digestion. ATP binding stimulates but is not required for WRN binding to this region. Thus, binding of WRN to the melted region underlies the efficient exonuclease activity directed at the nearby ends. In contrast, a WRN deletion mutant containing only the functional exonuclease domain does not detectably bind or degrade this substrate. These experiments indicate a bipartite structure and function for WRN, and we propose a model by which its DNA binding, helicase, and exonuclease activities function coordinately in DNA metabolism. These studies also suggest that partially unwound or noncomplementary regions of DNA could be physiological targets for WRN.

• Authors: Amrita Machwe, Liren Xiao, Shaji Theodore, David K Orren
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 277 Issue 6 Pg. 4492-504 (Feb 08 2002) ISSN: 0021-9258 [Print] United States
• PMID: 11717307 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• DNA-Binding Proteins
• Recombinant Proteins
• DNA
• Exodeoxyribonucleases
• Exonucleases
• Deoxyribonuclease I
• DNA Helicases
• RecQ Helicases
• WRN protein, human
• Werner Syndrome Helicase

Topics

• Base Sequence
• DNA (metabolism)
• DNA Footprinting
• DNA Helicases (metabolism)
• DNA-Binding Proteins (metabolism)
• Deoxyribonuclease I (metabolism)
• Exodeoxyribonucleases
• Exonucleases (metabolism)
• Humans
• Molecular Sequence Data
• RecQ Helicases
• Recombinant Proteins (metabolism)
• Werner Syndrome (metabolism)
• Werner Syndrome Helicase