RPA regulates telomerase action by providing Est1p access to chromosome ends

Schramke, Véra; Luciano, Pierre; Brevet, Vanessa; Guillot, Sylvine; Corda, Yves; Longhese, Maria Pia; Gilson, Éric; Géli, Vincent

doi:10.1038/ng1284

Cited by 140 publications

(131 citation statements)

References 37 publications

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“…Perhaps the Est1 DNA binding ability provides a means to telomere-associate independent of telomerase, but once telomerase arrives Est1 switches ligands and binds to TLC1-comparable DNA/RNA swapping has been previously postulated for other factors (22). Although the necessity of the replication protein A (RPA) for Est1 telomere loading suggests Est1 DNA binding activity is insufficient for telomere interactions (23). Alternatively Est1 DNA binding might be important for other non-telomeric cellular functions.…”

Section: Discussionmentioning

confidence: 99%

The conserved Est1 protein stimulates telomerase DNA extension activity

DeZwaan

Freeman

2009

Proc. Natl. Acad. Sci. U.S.A.

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The first telomerase cofactor identified was the budding yeast protein Est1, which is conserved through humans. While it is evident that Est1 is required for telomere DNA maintenance, understanding its mechanistic contributions to telomerase regulation has been limited. In vitro, the primary effect of Est1 is to activate telomerase-mediated DNA extension. Although Est1 displayed specific DNA and RNA binding, neither activity contributed significantly to telomerase stimulation. Rather Est1 mediated telomerase upregulation through direct contacts with the reverse transcriptase subunit. In addition to intrinsic Est1 functions, we found that Est1 cooperatively activated telomerase in conjunction with Cdc13 and that the combinatorial effect was dependent upon a known salt-bridge interaction between Est1 (K444) and Cdc13 (E252). Our studies provide insights into the molecular events used to control the enzymatic activity of the telomerase holoenzyme.telomere biology ͉ telomerase regulation

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Section: Discussionmentioning

confidence: 99%

The conserved Est1 protein stimulates telomerase DNA extension activity

DeZwaan

Freeman

2009

Proc. Natl. Acad. Sci. U.S.A.

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“…This would allow more rapid progression toward the final product, even if Dna2 binds the flap in the non-tracking mode. We have also considered that both Dna2 and RPA are proposed to be involved in overlapping cellular pathways, such as DNA repair, recombination, and telomere maintenance (20,(27)(28)(29)(45)(46)(47)(48). Dissociation of RPA by Dna2 could extend to these other pathways in which RPA removal is necessary for further processing of the DNA.…”

Section: Discussionmentioning

confidence: 99%

Dynamic Removal of Replication Protein A by Dna2 Facilitates Primer Cleavage during Okazaki Fragment Processing in Saccharomyces cerevisiae

Stewart

Miller

Campbell

et al. 2008

Journal of Biological Chemistry

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Eukaryotic Okazaki fragments are initiated by a RNA/DNA primer, which is removed before the fragments are joined. Polymerase ␦ displaces the primer into a flap for processing. Dna2 nuclease/helicase and flap endonuclease 1 (FEN1) are proposed to cleave the flap. The single-stranded DNA-binding protein, replication protein A (RPA), governs cleavage activity. Flap-bound RPA inhibits FEN1. This necessitates cleavage by Dna2, which is stimulated by RPA. FEN1 then cuts the remaining RPA-free flap to create a nick for ligation. Cleavage by Dna2 requires that it enter the 5-end and track down the flap. Because Dna2 cleaves the RPA-bound flap, we investigated the mechanism by which Dna2 accesses the protein-coated flap for cleavage. Using a nuclease-defective Dna2 mutant, we showed that just binding of Dna2 dissociates the flap-bound RPA. Facile dissociation is specific to substrates with a genuine flap, and will not occur with an RPA-coated single strand. We also compared the cleavage patterns of Dna2 with and without RPA to better define RPA stimulation of Dna2. Stimulation derived from removal of DNA folding in the flap. Apparently, coordinated with its dissociation, RPA relinquishes the flap to Dna2 for tracking in a way that does not allow flap structure to reform. We also found that RPA strand melting activity promotes excessive flap elongation, but it is suppressed by Dna2-promoted RPA dissociation. Overall, results indicate that Dna2 and RPA coordinate their functions for efficient flap cleavage and preparation for FEN1.Eukaryotic DNA replication involves synthesis of a leading strand in the direction of parental DNA unwinding, and because of the anti-parallel structure of DNA, a lagging strand in the opposite direction of unwinding. Although leading strand synthesis occurs continuously, the lagging strand must be synthesized in a discontinuous fashion. Lagging strand synthesis is initiated by the polymerase (pol) 3 ␣-primase complex, which synthesizes 8 -12 nucleotides (nt) of RNA followed by ϳ20-nt of DNA (1, 2). The pol ␣-primase complex is then replaced by a complex of the toroidal sliding clamp proliferating cell nuclear antigen and pol ␦, which is loaded onto the DNA by the clamp loader replication factor C. This complex synthesizes another 100 -150 nt of DNA before the primer terminus encounters the previously synthesized downstream segment. These segments, known as Okazaki fragments, must then be processed to create a continuous strand of DNA. Because pol ␣ does not possess proofreading activity and synthesizes with relatively low fidelity compared with pol ␦, it is proposed that the entire RNA/DNA primer, laid down by pol ␣, is removed to maintain genome integrity (3). Processing of the RNA/DNA primer is initiated when strand displacement synthesis, by pol ␦, raises the primer into a single-stranded (ss) flap intermediate (4 -6). Removal of this flap intermediate followed by fragment joining is known as Okazaki fragment processing.Several modes of Okazaki fragment processing have been proposed to occur ...

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“…RPA also has a role in maintenance of telomeres (7,12) and in regulation of the cell cycle (13,14). The common feature of all of these processes is that each has ssDNA intermediates that must be recognized and processed appropriately.…”

mentioning

confidence: 99%

Cellular Functions of Human RPA1

Haring

Mason

Binz³

et al. 2008

Journal of Biological Chemistry

102

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In eukaryotes, the single strand DNA (ssDNA)-binding protein, replication protein A (RPA), is essential for DNA replication, repair, and recombination. RPA is composed of the following three subunits: RPA1, RPA2, and RPA3. The RPA1 subunit contains four structurally related domains and is responsible for high affinity ssDNA binding. This study uses a depletion/replacement strategy in human cells to reveal the contributions of each domain to RPA cellular functions. Mutations that substantially decrease ssDNA binding activity do not necessarily disrupt cellular RPA function. Conversely, mutations that only slightly affect ssDNA binding can dramatically affect cellular function. The N terminus of RPA1 is not necessary for DNA replication in the cell; however, this region is important for the cellular response to DNA damage. Highly conserved aromatic residues in the high affinity ssDNA-binding domains are essential for DNA repair and cell cycle progression. Our findings suggest that as long as a threshold of RPA-ssDNA binding activity is met, DNA replication can occur and that an RPA activity separate from ssDNA binding is essential for function in DNA repair.Cell survival and proliferation depend on the efficient maintenance of genetic information. Human cells must accurately replicate billions of base pairs of DNA and identify and repair a wide variety of DNA lesions. One of the proteins required for the maintenance of genomic integrity is replication protein A (RPA). 3 RPA is a heterotrimeric single strand DNA (ssDNA)-binding protein, composed of 70-, 32-, and 14-kDa subunits, commonly referred to as RPA1, RPA2, and RPA3, respectively (1-3). Homologs of the three RPA subunits have been found in all eukaryotes examined (1, 4). The major biochemical activity of RPA is ssDNA binding; RPA can bind ssDNA with subnanomolar affinity (5). The ability of RPA to bind ssDNA is not sequence-specific; however, RPA has a higher affinity for pyrimidine-rich and telomeric ssDNA (6 -8).Replication protein A was originally isolated as a factor essential for in vitro DNA replication of simian virus 40 (SV40) (9 -11), and it has since been shown to be essential for a number of other DNA metabolic processes, including chromosomal DNA replication, repair, and recombination (1-3). RPA also has a role in maintenance of telomeres (7, 12) and in regulation of the cell cycle (13,14). The common feature of all of these processes is that each has ssDNA intermediates that must be recognized and processed appropriately. RPA interacts with ssDNA in the cell and with a number of proteins involved in these processes (3,15).Each of the RPA subunits has at least one domain containing an oligonucleotide/oligosaccharide-binding (OB) fold. This fold is found in many ssDNA-and sugar-binding proteins (16). The OB-folds in RPA are commonly referred to as DNA-binding domains (DBDs). RPA1 contains four OB-folds, and RPA2 and RPA3 have one OB-fold each. Although each OB-fold is structurally similar, the majority of the ssDNA binding occurs through two OB-fo...

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RPA regulates telomerase action by providing Est1p access to chromosome ends

Cited by 140 publications

References 37 publications

The conserved Est1 protein stimulates telomerase DNA extension activity

The conserved Est1 protein stimulates telomerase DNA extension activity

Dynamic Removal of Replication Protein A by Dna2 Facilitates Primer Cleavage during Okazaki Fragment Processing in Saccharomyces cerevisiae

Cellular Functions of Human RPA1

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