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.Telomeres, the nucleoprotein complexes at the ends of eukaryotic linear chromosomes, serve a number of vital cellular functions. Telomere capping protects the chromosome ends from nucleolytic degradation and provides a mechanism for cells to distinguish natural from broken ends, which signal DNA damage and are substrates for DNA repair processes (reviewed in references 7 and 13). The DNA component of telomeres is composed of tandem, simple repeats (TTAGGG in mammals), terminating with a 3Ј-protruding single strand of the guanosine-rich strand. Due to lack of a template to replicate the 3Ј overhang, most human somatic cells lose terminal DNA with each division; thus, telomeres also provide a buffer between the ends and the more internally located coding region of the genome (12,19). In germ line cells and some cells in highly proliferative tissues, telomere loss is counterbalanced through the activities of the ribonucleoprotein telomerase. In vitro assays reveal that catalytically active telomerase is minimally composed of an RNA (TER) and a protein (TERT) subunit (5,38,39). Telomerase adds telomeric repeats by iteratively reverse transcribing the template portion of its RNA subunit, using the 3Ј single-strand telomeric overhang as a primer (18, 42; reviewed in references 20 and 22).Proteins that specifically bind the telomere single-strand overhang have been identified in numerous organisms. The 3Ј telomeric overhang of the ciliate Oxytricha nova is bound by OnTEBP, a heterodimeric end-binding protein composed of an ␣ subunit and a ␤ subunit (14, 17). Crystal structures reveal that the ␣ subunit contains three oligonucleotide/oligosaccharide binding (OB) folds: the first two OB folds bind telomeric DNA with sequence specificity, and the third participates in protein-protein interactions with the ␤ subunit (21). A search for homologs of the OnTEBP ␣ subunit identified the Pot1 protein in Schizosaccharomyces pombe and human based on a weak sequence similarity with the first OB fold in the ␣ subunit (3). Genetic studies with S. pombe demonstrate a role for S. pombe POT1 in telomere end protection, since deletion of the pot1 gene results in the rapid loss of ...

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The RNase H activity of reverse transcriptase (RT) is presumably required to cleave the RNA genome following minus strand synthesis to free the DNA for use as a template during plus strand synthesis. However, since RNA degradation by RNase H appears to generate RNA fragments too large to spontaneously dissociate from the minus strand, we have investigated the possibility that RNA displacement by RT during plus strand synthesis contributes to the removal of RNA fragments. By using an RNase H ؊ mutant of Moloney murine leukemia virus (M-MuLV) RT, we demonstrate that the polymerase can displace long regions of RNA in hybrid duplex with DNA but that this activity is approximately 5-fold slower than DNA displacement and 20-fold slower than non-displacement synthesis. Furthermore, we find that although certain hybrid sequences seem nearly refractory to the initiation of RNA displacement, the same sequences may not significantly impede synthesis when preceded by a single-stranded gap. We find that the rate of RNA displacement synthesis by wild-type M-MuLV RT is significantly greater than that of the RNase H ؊ RT but remains less than the rate of non-displacement synthesis. M-MuLV nucleocapsid protein increases the rates of RNA and DNA displacement synthesis approximately 2-fold, and this activity appears to require the zinc finger domain.Retroviral replication requires the single-stranded RNA genome of the virus to be converted into double-stranded DNA through a complex series of reactions termed reverse transcription. This process appears to be catalyzed solely by the viral reverse transcriptase (RT) 1 that possesses the following two distinct enzymatic activities: a polymerase activity that synthesizes DNA using either RNA or DNA templates, and an RNase H activity that cleaves RNA in hybrid duplex with DNA (1, 2).The current model of reverse transcription proposes that the RNase H activity of RT is critical for several steps including degradation of the 5Ј end of the RNA genome following minusstrong-stop DNA synthesis to facilitate the first jump, specific cleavage at the polypurine tract to create the plus strand primer, and removal of the plus and minus strand primers (3). Additionally, it is presumed that the RNase H activity is required to degrade the RNA genome following minus strand synthesis to free the minus strand DNA for use as a template during plus strand synthesis (reviewed in Ref. 3). In vitro studies, however, suggest that RNA fragments that are too large to spontaneously dissociate from the minus strand remain following cleavage of the genome (4 -11). Furthermore, evidence that plus strand synthesis in several retroviral systems is discontinuous demonstrates that stably annealed RNA fragments persist in vivo (3,(12)(13)(14). This raises the interesting possibility that reverse transcription requires a mechanism for the displacement of genomic RNA fragments during plus strand synthesis.Most replicative polymerases require accessory proteins such as helicases and single-strand binding proteins (SSBs) ...

The ability of reverse transcriptase to generate, extend, and remove the primer derived from the polypurine tract (PPT) is vital for reverse transcription, since this process determines one of the ends required for integration of the viral DNA. Based on the ability of the RNase H activity of Moloney murine leukemia virus reverse transcriptase to cleave a long RNA/DNA hybrid containing the PPT, it appears that cleavages that could generate the plus-strand primer can occur by an internal cleavage mechanism without any positioning by an RNA 5-end, and such cleavages may serve to minimize cleavage events within the PPT itself. If the PPT were to be cleaved inappropriately just upstream of the normal plus-strand origin site, the resulting 3-ends would not be extended by reverse transcriptase. Extension of the PPT primer by at least 2 nucleotides is sufficient for recognition and correct cleavage by RNase H at the RNA-DNA junction to remove the primer. Specific removal of the PPT primer after polymerase extension deviates from the general observation that primer removal occurs by cleavage one nucleotide away from the RNA-DNA junction and suggests that the same PPT specificity determinants responsible for generation of the PPT primer also direct PPT primer removal. Once the PPT primer has been extended and removed from the nascent plus-strand DNA, reinitiation at the resulting plus-strand primer terminus does not occur, providing a mechanism to prevent the repeated initiation of plus strands.Reverse transcriptase converts the single-stranded retroviral RNA genome into the linear double-stranded DNA that integrates into the chromosome of a host cell (1, 2). The reverse transcriptase of Moloney murine leukemia virus (MMLV) 1 is a 75-kDa protein that contains an NH 2 -terminal DNA-and RNAdependent DNA polymerase activity and a COOH-terminal RNase H activity. Although the polymerase and RNase H activities are functionally separable (3-6), the polymerase and RNase H domains function in an interdependent manner (3, 7-10). The polymerase activity extends both RNA and DNA primers, although efficient extension from RNA primers appears limited to the host cell-derived tRNA primer used for minus-strand DNA synthesis and the primer used for plussense DNA synthesis that is derived from the polypurine tract (PPT) sequence in the viral genome (reviewed in Refs. 2 and 11) (12-20). The RNase H activity acts primarily as an endonuclease, hydrolyzing the RNA in an RNA/DNA hybrid to produce 3Ј-hydroxyl and 5Ј-phosphate ends (11,21,22). Cleavage by the RNase H activity of reverse transcriptase specifically generates the PPT primer during the process of reverse transcription (15,(23)(24)(25)(26). RNase H is also responsible for removing the tRNA and PPT primers from the nascent DNA strands after they have been extended and for general degradation of the viral genome after minus-strand DNA synthesis (reviewed in Ref. 11) (12,15,24,[27][28][29][30].Previous studies have indicated that two different modes of RNase H activity can be distingui...

During reverse transcription, the RNase H activity of reverse transcriptase specifically cleaves the viral genome within the polypurine tract (PPT) to create the primer used for the initiation of plus-strand DNA synthesis and nonspecifically cleaves the viral genome to facilitate synthesis of plus-strand DNA. To understand how primer length and sequence affect generation and utilization of the PPT, we employed short hybrid substrates containing or lacking the PPT to evaluate cleavage, extension, and binding by reverse transcriptase. Substrates containing RNAs with the correct 3 end for initiation of plus-strand synthesis were extended equally well by reverse transcriptase, but primer length affected susceptibility to RNase H cleavage. RNA substrates with 3 ends extending beyond the plus-strand initiation site were extended poorly but were specifically cleaved to generate the correct 3 end for initiation of plus-strand synthesis. Substrates containing RNAs lacking the PPT were cleaved nonspecifically and extended inefficiently. Specific cleavages to generate the plus-strand primer and 5-end-directed cleavages were kinetically favored over cleavages that destroyed the PPT primer or degraded other short RNA fragments. The PPT was not intrinsically resistant to cleavage by the isolated RNase H domain, and the isolated polymerase domain extended RNA primers containing the PPT sequence irrespective of the primer 3 end. These results provide insights into how reverse transcriptase generates and selectively utilizes the PPT primer for initiation of plus-strand DNA synthesis. Moloney murine leukemia virus (M-MuLV)1 converts its single-stranded plus-sense RNA genome into a double-stranded DNA molecule through the replicative process termed reverse transcription (reviewed in Ref. 1). Minus-strand DNA synthesis initiates from a host cell-derived tRNA primer and extends through a unique sequence (U5) and a terminal repeat sequence (R) at the 5Ј end of the genome before carrying out the first template jump to a second R sequence found at the 3Ј end of the viral genome. As minus-sense DNA synthesis progresses, a purine-rich sequence in the RNA genome termed the polypurine tract (PPT) that lies immediately adjacent to a downstream unique sequence (U3) is copied and subsequently cleaved to generate the PPT primer. This primer is used to initiate plus-strand DNA synthesis, which extends through U3-R-U5 and continues after a second jump to the 5Ј end of the DNA template. Displacement synthesis is required to complete duplex DNA synthesis and formation of the redundant DNA ends called long terminal repeats (LTRs) (2).The multifunctional enzyme that carries out this replicative process is the virally encoded reverse transcriptase (1). For M-MuLV, reverse transcriptase is a 75-kDa polypeptide that contains an amino-terminal RNA-and DNA-dependent DNA polymerase activity and a carboxyl-terminal RNase H activity that cleaves the RNA portion of an RNA/DNA hybrid. The RNase H and polymerase activities of reverse transcriptase are separabl...