Telomerase Activity and Telomere Length in Daphnia

May Telomerase Activity and Telomere Length in Daphnia Charles Schumpert 0 1 2 Jacob Nelson 0 1 2 Eunsuk Kim 0 1 2 Jeffry L. Dudycha 0 1 2 Rekha C. Patel 0 1 2 0 Department of Biological Sciences, University of South Carolina , Columbia, South Carolina , United States of America 1 Funding: This work was supported by National Institutes of Health grant 1R01AG037969-01 awarded to JLD and RCP. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript 2 Academic Editor: Gabriele Saretzki, University of Newcastle, UNITED KINGDOM Telomeres, comprised of short repetitive sequences, are essential for genome stability and have been studied in relation to cellular senescence and aging. Telomerase, the enzyme that adds telomeric repeats to chromosome ends, is essential for maintaining the overall telomere length. A lack of telomerase activity in mammalian somatic cells results in progressive shortening of telomeres with each cellular replication event. Mammals exhibit high rates of cell proliferation during embryonic and juvenile stages but very little somatic cell proliferation occurs during adult and senescent stages. The telomere hypothesis of cellular aging states that telomeres serve as an internal mitotic clock and telomere length erosion leads to cellular senescence and eventual cell death. In this report, we have examined telomerase activity, processivity, and telomere length in Daphnia, an organism that grows continuously throughout its life. Similar to insects, Daphnia telomeric repeat sequence was determined to be TTAGG and telomerase products with five-nucleotide periodicity were generated in the telomerase activity assay. We investigated telomerase function and telomere lengths in two closely related ecotypes of Daphnia with divergent lifespans, short-lived D. pulex and long-lived D. pulicaria. Our results indicate that there is no age-dependent decline in telomere length, telomerase activity, or processivity in short-lived D. pulex. On the contrary, a significant age dependent decline in telomere length, telomerase activity and processivity is observed during life span in long-lived D. pulicaria. While providing the first report on characterization of Daphnia telomeres and telomerase activity, our results also indicate that mechanisms other than telomere shortening may be responsible for the strikingly short life span of D. pulex. - Competing Interests: The authors have declared that no competing interests exist. Introduction Telomeres, the ends of linear chromosomes, have been studied extensively in relation to cellular aging and senescence [1,2,3]. Composed of repetitive nucleotide sequences (TTAGGG for mammals) associated with proteins, telomeres protect important genetic information of linear chromosomes from deletion arising due to the end replication problem [1,3]. The process of DNA replication leads to progressive shortening of linear chromosomes at the telomeres due to the fact that DNA polymerases can only polymerize in a 5 to 3 direction and require a primer with a free 3-OH group [1,3]. This inability to replicate linear DNA on the lagging strands all the way to ends necessitates the telomerase, an enzyme responsible for de novo addition of telomeric repeats to chromosomal ends [4]. Telomerase is a ribonucleoprotein complex, comprised of a protein catalytic subunit TERT (Telomeric Reverse Transcriptase), and an RNA template termed TERC (telomeric RNA Component) [1,3]. Telomerase activity is essential for maintaining telomere length throughout cellular lifespan. Early in human development, telomerase is constitutively active in cells but after birth it is active predominately in stem cells and germ cells with most somatic tissues having no telomerase activity [5,6]. Each individual DNA replication event of human telomerase-negative somatic cells leads to a loss of 100 bp of telomeric sequence, resulting in a progressive decline in telomere length with each cellular division [7]. Because of this progressive telomere shortening, human somatic cells can only undergo approximately 50 to 80 cellular replication events before becoming senescent [7]. Thus, telomere length is essential for normal cellular function and proliferation as well as chromosome stability. In the absence of proper telomere complex formation, the double-stranded break repair pathway can be initiated resulting in apoptosis or senescence [8,9]. Thus, telomeres serve a protective molecular role by shielding the loss of important genetic information as well as by maintaining chromosome stability throughout the cellular lifespan. Telomerase has also been implicated in nuclear DNA damage repair and plays a protective role for mitochondrial DNA during oxidative stress response during which telomerase shuttles from the nucleus to the mitochondria [10,11,12,13,14,15]. In this study we investigated telomerase activity, the telomeric repeat sequence, and telomere lengths in Daphnia, a freshwater crustacean, and an emerging model in aging liuresearch. Daphnia has been used extensively as a model in ecotoxicology studies [16] and with a fully sequenced genome of D. pulex, it is an emerging model in biomedical research [17,18]. Two ecotypes of Daphnia are of interest in relation to aging, D. pulex and D. pulicaria [19,20,21,22,23,24]. D. pulex is found in small transitory ponds, in which selection favors short longevity due to the limited time the ponds have water. In a laboratory environment D. pulex exhibits a lifespan on average of about 20 days [20,23]. D. pulicaria lives primarily in a stable environment of stratified lakes that are present all year long. In the lab D. pulicaria exhibits lifespans on average of about 70 days [20,23]. Genetically, the two ecotypes are almost identical and are capable of interbreeding with viable offspring in the wild [21]. Daphnia can be easily cultured and undergo cyclic parthenogenduesis [16], thus enabling creation of clonal lineages without the genetic variation normally associated with sexually reproducing organisms. Being crustaceans, Daphnia constantly shed their outer carapace and have regenerative cellular capacities [16]. Due to these unique characteristics Daphnia is a interesting model organism for understanding cellular processes associated with of aging. We present characterization of the telomere length, telomerase activity and processivity in the two ecotypes, the short-lived D. pulex and the long lived D. pulicaria. In the short-lived D. pulex, telomere length did not decline with age; however, in the long-lived D. pulicaria, telomere length decreased with age. Accordingly, telomerase activity in D. pulex is relatively constant throughout the life span, whereas in D. pulicaria, it declines considerably with age. In addition, the telomerase processivity increased with age in D. pulex, whereas in D. pulicaria it declined with age. This is an important initial study to investiga (...truncated)