Functional dissection of human and mouse POT1 proteins

The single-stranded telomeric DNA binding protein POT1 protects mammalian chromosome ends from the ATR-dependent DNA damage response, regulates telomerase-mediated telomere extension, and limits 5'-end resection at telomere termini. Whereas most mammals have a single POT1 gene, mice have two POT1 proteins that are functionally distinct. POT1a represses the DNA damage response, and POT1b controls 5'-end resection. In contrast, as we report here, POT1a and POT1b do not differ in their ability to repress telomere recombination. By swapping domains, we show that the DNA binding domain of POT1a specifies its ability to repress the DNA damage response. However, no differences were detected in the in vitro DNA binding features of POT1a and POT1b. In contrast to the repression of ATR signaling by POT1a, the ability of POT1b to control 5'-end resection was found to require two regions in the C terminus, one corresponding to the TPP1 binding domain and a second representing a new domain located between amino acids (aa) 300 and 350. Interestingly, the DNA binding domain of human POT1 can replace that of POT1a to repress ATR signaling, and the POT1b region from aa 300 to 350 required for the regulation of the telomere terminus is functionally conserved in human POT1. Thus, human POT1 combines the features of POT1a and POT1b.     

Protect and regulate: Recent findings on plant POT1-like proteins

Single-stranded DNA-binding proteins form protective caps at the chromosome ends and their binding is important in the regulation of telomerase access to telomeres. This group of proteins is represented by POT1 proteins described in yeast, humans and other model organisms. Here we review recent findings obtained in Arabidopsis POT1-like paralogs, namely the observed diversity in their interaction features and corresponding functions.     

POT1 proteins in green algae and land plants: DNA-binding properties and evidence of co-evolution with telomeric DNA

Telomeric DNA terminates with a single-stranded 3′ G-overhang that in vertebrates and fission yeast is bound by POT1 (Protection Of Telomeres). However, no in vitro telomeric DNA binding is associated with Arabidopsis POT1 paralogs. To further investigate POT1–DNA interaction in plants, we cloned POT1 genes from 11 plant species representing major branches of plant kingdom. Telomeric DNA binding was associated with POT1 proteins from the green alga Ostreococcus lucimarinus and two flowering plants, maize and Asparagus. Site-directed mutagenesis revealed that several residues critical for telomeric DNA recognition in vertebrates are functionally conserved in plant POT1 proteins. However, the plant proteins varied in their minimal DNA-binding sites and nucleotide recognition properties. Green alga POT1 exhibited a strong preference for the canonical plant telomere repeat sequence TTTAGGG with no detectable binding to hexanucleotide telomere repeat TTAGGG found in vertebrates and some plants, including Asparagus. In contrast, POT1 proteins from maize and Asparagus bound TTAGGG repeats with only slightly reduced affinity relative to the TTTAGGG sequence. We conclude that the nucleic acid binding site in plant POT1 proteins is evolving rapidly, and that the recent acquisition of TTAGGG telomere repeats in Asparagus appears to have co-evolved with changes in POT1 DNA sequence recognition.     

Two telomeric ends of acrocentric chromosome play distinct roles in homologous chromosome synapsis in the fetal mouse oocyte

Chromosoma - In mammalian oocytes, proper chromosome segregation at the first meiotic division is dictated by the presence and site of homologous chromosome recombination, which takes place in...     

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.     

Maintenance of very long telomeres by recombination in the Kluyveromyces lactis stn1-M1 mutant involves extreme telomeric turnover, telomeric circles, and concerted telomeric amplification

Some cancers utilize the recombination-dependent process of alternative lengthening of telomeres (ALT) to maintain long heterogeneous telomeres. Here, we studied the recombinational telomere elongation (RTE) of the Kluyveromyces lactis stn1-M1 mutant. We found that the total amount of the abundant telomeric DNA in stn1-M1 cells is subject to rapid variation and that it is likely to be primarily extrachromosomal. Rad50 and Rad51, known to be required for different RTE pathways in Saccharomyces cerevisiae, were not essential for the production of either long telomeres or telomeric circles in stn1-M1 cells. Circles of DNA containing telomeric repeats (t-circles) either present at the point of establishment of long telomeres or introduced later into stn1-M1 cells each led to the formation of long tandem arrays of the t-circle's sequence, which were incorporated at multiple telomeres. These tandem arrays were extraordinarily unstable and showed evidence of repeated rounds of concerted amplification. Our results suggest that the maintenance of telomeres in the stn1-M1 mutant involves extreme turnover of telomeric sequences from processes including both large deletions and the copying of t-circles.     

Human protection of telomeres 1 (POT1) is a negative regulator of telomerase activity in vitro

The telomeric single-strand DNA binding protein protection of telomeres 1 (POT1) protects telomeres from rapid degradation in Schizosaccharomyces pombe and has been implicated in positive and negative telomere length regulation in humans. Human POT1 appears to interact with telomeres both through direct binding to the 3' overhanging G-strand DNA and through interaction with the TRF1 duplex telomere DNA binding complex. The influence of POT1 on telomerase activity has not been studied at the molecular level. We show here that POT1 negatively effects telomerase activity in vitro. We find that the DNA binding activity of POT1 is required for telomerase inhibition. Furthermore, POT1 is incapable of inhibiting telomeric repeat addition to substrate primers that are defective for POT1 binding, suggesting that in vivo, POT1 likely affects substrate access to telomerase.     

Two distinct quinone-modulated modes of antimycin-sensitive cytochrome b reduction in the cytochrome bc1 complex

Reduction of cytochrome b-560 (analogous to cyt b-562 of mitochondria) via an antimycin-sensitive route has been revealed in chromatophores of the photosynthetic bacterium, Rhodopseudomonas sphaeroides Ga. Indeed, the results suggest that two reductive mechanisms can be operative. One is consistent with the idea that the quinol generated at the reaction center QB site enters the Q pool and, via the Qc site, equilibrates with cytochrome b-560. The other reductive mode circumvents redox equilibrium with the pool; we consider that this could result from a direct encounter of the reaction center with the bc1 complex perhaps involving a direct QB-Qc site interaction. This latter reaction is suppressed by occupancy of the Qc site, not only by antimycin but by ubiquinol and ubiquinone.     

Expression characteristics of pokeweed antiviral proteins (PAPs): Two distinct types of proteins

Pokeweed antiviral proteins (PAPs) become novel therapeutic agents in relation to application in human viral diseases and cancer, as well as potent tools in plant system for defending viral infection. We have studied the expression characteristics of PAPs in pokeweed plants by western blot analysis. PAP-I was constitutively expressed in leaves, stems and roots of the pokeweed plant, while PAP-II was not expressed in roots. The expression of PAP-II began in May and then gradually increased with development of the plants. The PAP-II expression was induced and/or stimulated not only by biotic stresses, such as insect pests and viral infection, but also by abiotic stresses, like drought. Interestingly, low-light intensity was found to be more effective than high-light in the expression of both PAP-I and PAP-II. Our results suggest that PAP-II appears to have an additive effect in terms of protection of the plant against pathogens during summer-time when the plant actively grows and is attacked by various pathogens.     

How the human telomeric proteins TRF1 and TRF2 recognize telomeric DNA: a view from high-resolution crystal structures

Human telomeres consist of tandem arrays of TTAGGG sequence repeats that are specifically bound by two proteins, TRF1 and TRF2. They bind to DNA as preformed homodimers and have the same architecture in which the DNA-binding domains (Dbds) form independent structural units. Despite these similarities, TRF1 and TRF2 have different functions at telomeres. The X-ray crystal structures of both TRF1- and TRF2-Dbds in complex with telomeric DNA (2.0 and 1.8 angstroms resolution, respectively) show that they recognize the same TAGGGTT binding site by means of homeodomains, as does the yeast telomeric protein Rap1p. Two of the three G-C base pairs that characterize telomeric repeats are recognized specifically and an unusually large number of water molecules mediate protein-DNA interactions. The binding of the TRF2-Dbd to the DNA double helix shows no distortions that would account for the promotion of t-loops in which TRF2 has been implicated.     

Secretory proteins of the lung in rodents: immunocytochemistry

The reactivity of rabbit antisera to rat lung secretory proteins with other rodent species was evaluated by immunocytochemistry. Rabbit anti-rat surfactant apoprotein antiserum reacts with the cytoplasm of rat, mouse, and hamster type II pneumocytes and is specific for these cells. Rabbit antiserum to rat Clara cell secretory proteins stains rat, mouse, and hamster Clara cells. Rabbit antisera specific to the two antigenic types of rat Clara cell antigens were also both reactive with rat, mouse, and hamster Clara cells. An antiserum to the non-serum proteins of hamster lung lavage was also prepared and shown to be specifically reactive with hamster Clara cells. The availability of specific reagents for secretory proteins of rodent lungs is expected to facilitate studies of the respective cell types in various pathologic states.     

Retrofitting the genome: L1 extinction follows endogenous retroviral expansion in a group of muroid rodents

Long interspersed nuclear element 1 (LINE-1; L1) retrotransposons are the most common retroelements in mammalian genomes. Unlike individual families of endogenous retroviruses (ERVs), they have remained active throughout the mammalian radiation and are responsible for most of the retroelement movement and much genome rearrangement within mammals. They can be viewed as occupying a substantial niche within mammalian genomes. Our previous demonstration that L1s and B1 short interspersed nuclear elements (SINEs) are inactive in a group of South American rodents led us to ask if other elements have amplified to fill the empty niche. We identified a novel and highly active family of ERVs (mysTR). To determine whether loss of L1 activity was correlated with expansion of mysTR, we examined mysTR activity in four South American rodent species that have lost L1 and B1 activity and four sister species with active L1s. The copy number of recent mysTR insertions was extremely high, with an average of 4,200 copies per genome. High copy numbers exist in both L1-active and L1-extinct species, so the mysTR expansion appears to have preceded the loss of both SINE and L1 activity rather than to have filled an empty niche created by their loss. It may be coincidental that two unusual genomic events--loss of L1 activity and massive expansion of an ERV family--occur in the same group of mammals. Alternatively, it is possible that this large ERV expansion set the stage for L1 extinction.