The S. pombe protection of telomeres 1 (SpPot1) protein recognizes the 3′ single-stranded ends of telomeres and provides essential protective and regulatory functions. The ssDNA-binding activity of SpPot1 is conferred by its ssDNA-binding domain, Pot1-DBD (residues 1-389), which can be further separated into two distinct domains, Pot1pN (residues 1-187) and Pot1pC (residues 188-389).Here we show that Pot1pC, like Pot1pN, can function independently of Pot1-DBD and binds specifically to a minimal nonameric oligonucleotide, d(GGTTACGGT), with a K D of 400 +/-70 nM (specifically recognized nucleotides in bold). NMR chemical shift perturbation analysis indicates that the overall structures of the isolated Pot1pN and Pot1pC domains remain intact in Pot1-DBD. Furthermore, alanine scanning reveals modest differences in the ssDNA-binding contacts provided by isolated Pot1pN and within Pot1-DBD. Although the global character of both Pot1pN and Pot1pC is maintained in Pot1-DBD, chemical shift perturbation analysis highlights localized structural differences within the G1/G2 and T3/T4 binding pockets of Pot1pN in Pot1-DBD, which correlate with its distinct ssDNA-binding activity. Furthermore, we find evidence for a putative interdomain interface on Pot1pN that mediates interactions with Pot1pC that ultimately result in the altered ssDNA-binding activity of Pot1-DBD. Together, these data provide insight into the mechanisms underlying the activity and regulation of SpPot1 at the telomere. Keywordstelomeres; ssDNA-binding domain; end-protection; OB fold; Pot1Eukaryotic chromosomes terminate in a conserved 3′ single-stranded DNA overhang. If left unattended, this overhang triggers the activation of the DNA damage response leading to chromosomal abnormalities that halt cellular proliferation (1). This outcome is circumvented by the protective functions of specialized telomere-associated proteins collectively referred to as the telomere end-protection (TEP) family. In addition to their protective functions, TEP proteins also participate in numerous regulatory functions at telomeres, including controlling † We acknowledge the NRSA Postdoctoral Fellowship GM-071257 (to J.E.C.), National Institutes of Health Training Appointment (NIH) the nucleotide addition activity of telomerase (2-7), coordinating lagging (3′ → 5′) strand synthesis (8), fixing the termination point of lagging strand resection (9) and controlling the formation of higher order telomere structure (i.e., t-loops and G-quartet structures) (10,11). As a result of the critical nature of these regulatory activities, TEP proteins are essential for normal cellular proliferation and long-term survival.A universally shared feature of TEP proteins is their ability to specifically bind to the 3′ ssDNA ends of telomeres through a conserved ssDNA-binding domain (DBD). Structural and bioinformatics (12) Figure 1). This ssDNA-binding interface contains three distinct binding pockets that constrain the bound oligonucleotide in a highly compacted orientation: the G1/G2, T3...

Linear chromosomes terminate in specialized nucleoprotein structures called telomeres, which are required for genomic stability and cellular proliferation. Telomeres end in an unusual 3′ singlestrand overhang that requires a special capping mechanism to prevent inappropriate recognition by the DNA damage machinery. In Schizosaccharomyces pombe, this protective function is mediated by the Pot1 protein, which binds specifically and with high affinity to telomeric ssDNA. We have characterized the thermodynamics and accommodation of both cognate and noncognate telomeric single-stranded DNA (ssDNA) sequences by Pot1pN, an autonomous ssDNA-binding domain (residues 1-187) found in full-length S. pombe Pot1. Direct calorimetric measurements of cognate telomeric ssDNA binding to Pot1pN show favorable enthalpy, unfavorable entropy, and a negative heat-capacity change. Thermodynamic analysis of the binding of noncognate telomeric ssDNA to Pot1pN resulted in unexpected changes in free energy, enthalpy, and entropy. Chemical-shift perturbation and structural analysis of these bound noncognate sequences show that these thermodynamic changes result from the structural rearrangement of both Pot1pN and the bound oligonucleotide. These data suggest that the ssDNA-binding interface is highly dynamic and, in addition to the conformation observed in the crystal structure of the Pot1pN/d(GGTTAC) complex, capable of adopting alternative thermodynamically equivalent conformations.Protein recognition of nucleic acids is a key underlying molecular recognition event that guides replication, transcription, recombination, and other essential biological processes. To † We acknowledge the Wenner-Gren Foundation Postdoctoral Fellowship (to J.L.F.), National Research Service Award (NRSA) Postdoctoral Fellowship GM-071257 (to J.E.C.), and National Institutes of Health (NIH) GM-059414 (to D.S.W.), and National Science Foundation (NSF) (to D.S.W.) (MCB-0617956) for funding this research.© Copyright 2008 by the American Chemical Society * To whom correspondence should be addressed. date, a number of structural, biochemical, and thermodynamic studies have provided a fundamental understanding of how protein/double-stranded DNA (dsDNA) 1 recognition occurs. These studies reveal that dsDNA recognition is mediated by direct and indirect protein contacts that form intricate networks of hydrogen bonds with the bases (1-3). Thermodynamically, dsDNA recognition is generally characterized by negative enthalpy, positive entropy (ascribed to water/ion release), and large negative heat-capacity changes (ascribed to surface exclusion of the solvent) (4). Furthermore, the specific recognition of dsDNA typically leads to larger negative heat-capacity changes when compared to its nonspecific counterpart (1, 2). Although these characteristics are common, they can be modulated by many physical processes, including (1) conformational changes of the molecules upon binding (5), (2) redistribution of ion atmospheres, (3) dynamic behavior, and (4) hydration patte...

Previously we found that live, fresh or thawed boar sperm show little tendency to accumulate ROS spontaneously, but live sperm accumulated ROS during a 30-min incubation with xanthine and xanthine oxidase and showed marked reduction in motility. High mitochondrial transmembrane potential (MMP) is required to drive the F0/F1 ATPase responsible for producing ATP in most cell types, and ATP is required for sperm motility. This experiment was conducted to investigate the effects of menadione (disrupter of electron transport at Complex I) on sperm motility, MMP, and ATP content. The incidence of cells with high MMP was determined by measuring the fluorescence of JC-1 aggregates bound to the inner mitochondrial membrane using flow cytometry. Computer-assisted motion analysis was conducted using the IVOS version 12 (Hamilton Thorne Research, Beverly, MA, USA), and ATP (pmoles/106 sperm) was determined using the luciferin-luciferase assay. Sperm from six boars were individually Percoll washed to eliminate seminal plasma and incubated at 40 � 106/mL with 0, 1, 10, or 30 �M menadione for 5, 30, 60, and 120 min at 38�C in a modified Tyrode's medium containing 0.1% polyvinyl alcohol with no bicarbonate or BSA. The formation of ROS was confirmed by measuring the red fluorescence developed by the oxidation of hydroethidine to ethidium using flow cytometry. Whereas the basal level of ethidium fluorescence in the absence of menadione was low (2% ethidium-positive cells at 5 min), 10 and 30 �M menadione increased (P < 0.05) the percentage of ethidium-positive cells to 47 and 87%, respectively, at 30 min. Sperm motility did not decrease significantly (79-83%) during the 120 min incubation with no menadione, but menadione caused a significant dose-related decrease (P < 0.05) over time, with 10 and 30 �M menadione decreasing motility to 60 and 40%, 51 and 7%, and 10 and 1% at 30, 60, and 120 min, respectively. JC-1 aggregate fluorescence intensity decreased (P < 0.05) by 45-60% in a dose-related fashion at 120 min compared to the same doses at 5 min. Sperm viability, as measured by number of propidium iodide negative cells, averaged 93.6% during the incubation and was not significantly affected by treatment. The effect of menadione on ATP content was much less than that on motility or JC-1 fluorescence intensity. Mean ATP content averaged 63 pmoles through 60 min at all menadione doses; at 120 min only 30 �M menadione decreased (P < 0.05) ATP to 43 pmoles, compared to all other treatments. Menadione caused an increase in ROS formation and a decline in MMP, which suggested uncoupling of the respiratory chain and oxidative phosphorylation. However, sperm ATP content was not highly correlated with motility. This suggests that ATP content was maintained by the activity of other intermediary metabolism pathways. The reduction in motility may have been due to a ROS induced lesion in ATP utilization or in the contractile apparatus of the cell.