Nucleic Acids Research Advance Access originally published online on June 25, 2008
Nucleic Acids Research 2008 36(13):4242-4256; doi:10.1093/nar/gkn385
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Nucleic Acids Research, 2008, Vol. 36, No. 13 4242-4256
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Nucleic Acid Enzymes |
Mechanism and substrate specificity of telomeric protein POT1 stimulation of the Werner syndrome helicase
1Department of Environmental and Occupational Health, University of Pittsburgh Graduate School of Public Health, Pittsburgh, PA 15219 and 2Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109-0606, USA
*To whom correspondence should be addressed. Tel: +1 412 521 3034; Fax: +1 412 624 9361; Email: plo4{at}pitt.edu
Received April 28, 2008. Revised May 15, 2008. Accepted May 31, 2008.
Loss of the RecQ helicase WRN protein causes the cancer-prone progeroid disorder Werner syndrome (WS). WS cells exhibit defects in DNA replication and telomere preservation. The telomeric single-stranded binding protein POT1 stimulates WRN helicase to unwind longer telomeric duplexes that are otherwise poorly unwound. We reasoned that stimulation might occur by POT1 recruiting and retaining WRN on telomeric substrates during unwinding and/or by POT1 loading on partially unwound ssDNA strands to prevent strand re-annealing. To test these possibilities, we used substrates with POT1-binding sequences in the single-stranded tail, duplex or both. POT1 binding to ssDNA tails did not alter WRN activity on nontelomeric duplexes or recruit WRN to telomeric ssDNA. However, POT1 bound tails inhibited WRN activity on telomeric duplexes with a single 3'-ssDNA tail, which mimic telomeric ends in the open conformation. In contrast, POT1 bound tails stimulated WRN unwinding of forked telomeric duplexes. This indicates that POT1 interaction with the ssDNA/dsDNA junction regulates WRN activity. Furthermore, POT1 did not enhance retention of WRN on telomeric forks during unwinding. Collectively, these data suggest POT1 promotes the apparent processivity of WRN helicase by maintaining partially unwound strands in a melted state, rather than preventing WRN dissociation from the substrate.
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