Mechanism and substrate specificity of telomeric protein POT1 stimulation of the Werner syndrome helicase.
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.
Sowd
Gregory
G
Department of Environmental and Occupational Health, University of Pittsburgh Graduate School of Public Health, Pittsburgh, PA 15219, USA.
Lei
Ming
M
Opresko
Patricia L
PL
D004247:DNA / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004789:Enzyme Activation; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D006801:Humans; D053484:RecQ Helicases / Q000378:metabolism*; D000089804:Shelterin Complex; D013045:Species Specificity; D013379:Substrate Specificity; D016615:Telomere / Q000737:chemistry / Q000378:metabolism; D034501:Telomere-Binding Proteins / Q000378:metabolism*; D000071657:Werner Syndrome Helicase
D004277:DNA, Single-Stranded; C440849:POT1 protein, human; D000089804:Shelterin Complex; D034501:Telomere-Binding Proteins; D004247:DNA; D005090:Exodeoxyribonucleases; D053484:RecQ Helicases; C098785:WRN protein, human; D000071657:Werner Syndrome Helicase