Abstract
Werner syndrome is an inherited human progeriod syndrome caused by mutations in the gene encoding the Werner Syndrome protein, WRN. It has both 3'-5' DNA helicase and exonuclease activities, and is suggested to have roles in many aspects of DNA metabolism, including DNA repair and telomere maintenance. The DNA-PK complex also functions in both DNA double strand break repair and telomere maintenance. Interaction between WRN and the DNA-PK complex has been reported in DNA double strand break repair, but their possible cooperation at telomeres has not been reported. This study analyzes thein vitro and in vivo interaction at the telomere between WRN and DNA-PKcs, the catalytic subunit of DNA-PK. The results show that DNA-PKcs selectively stimulates WRN helicase but not WRN exonuclease in vitro, affecting that WRN helicase unwinds and promotes the release of the full-length invading strand of a telomere D-loop model substrate. In addition, the length of telomeric G-tails decreases in DNA-PKcs knockdown cells, and this phenotype is reversed by overexpression of WRN helicase. These results suggest that WRN and DNA-PKcs may cooperatively prevent G-tail shortening in vivo.
Highlights
Werner syndrome (WS) is a hereditary disorder associated with symptoms of premature aging, including early onset of cataracts, osteoporosis, atherosclerosis and cancer [1, 2]
The melted region and the invading ssDNA carry telomeric repeats, such that the DNA substrate mimics a telomeric D-loop. Previous studies with this DNA substrate showed that Werner syndrome protein (WRN) exonuclease partially degrades and the WRN helicase unwinds and releases the INV DNA strand, which is stable after release because WRN exonuclease does not efficiently degrade ssDNA [12]
The total ssDNA product was similar in WRN reactions with or without DNA-PKcs (Figures 1B and 1C, lanes 6). These results suggested two possibilities; i) the processivity of WRN exonuclease is inhibited by DNA-PKcs, or ii) the processivity of WRN helicase is stimulated by DNAPKcs
Summary
Werner syndrome (WS) is a hereditary disorder associated with symptoms of premature aging, including early onset of cataracts, osteoporosis, atherosclerosis and cancer [1, 2]. The DNA-PK complex, which is composed of a catalytic subunit, DNA-PKcs, regulatory subunits Ku70, and Ku80, is a DNA damage sensing serine-threonine protein kinase that is critical for repair of DNA double strand breaks. This complex was found at telomeres and DNA-PKcs-deficient cells exhibit dysfunctional telomeres [18, 19]. In addition to the similar defects of telomere in WS cells and DNA-PKcs deficient cells, DNA-PKcs interacts with and phosphorylates WRN in response to DNA double-strand breaks [20,21,22] These two proteins may cooperate in telomere metabolism
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