Human replication protein A unfolds telomeric G-quadruplexes

Salas, Tonatiuh Romero; Petruseva, I. O.; Lavrik, Olga I.; Bourdoncle, Anne; Mergny, Jean‐Louis; Favre, Alain; Saintomé, Carole

doi:10.1093/nar/gkl564

Cited by 168 publications

(161 citation statements)

References 51 publications

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“…S9). It is also possible that v5 competes and/or cooperates with the other single-stranded telomere-binding proteins, such as RPA (41) and hnRNPs (42), to exert its telomere-protective activity. Although the exact molecular mechanism of telomere protection by v5 still needs to be clarified, the central region present in the telomere-protective v5, but not in the nonprotective v2, should play a critical role in interacting with and/or regulating other factors yet to be identified.…”

Section: Cancer Researchmentioning

confidence: 99%

Functional Diversity of Human Protection of Telomeres 1 Isoforms in Telomere Protection and Cellular Senescence

et al. 2007

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Protection of telomeres 1 (POT1) proteins in various organisms bind telomeres and regulate their structure and function. In contrast to mice carrying two distinct POT1 genes encoding two POT1 proteins (POT1a and POT1b), humans have the single POT1 gene. In addition to full-length POT1 protein (variant v1), the human POT1 gene encodes four other variants due to alternative RNA splicing (variants v2, v3, v4, and v5), whose functions are poorly understood. The functional analyses of the NH 2 -terminally and COOH-terminally truncated POT1 variants in this study showed that neither the single-stranded telomere-binding ability of the NH 2 -terminal oligonucleotide-binding (OB) folds nor the telomerasedependent telomere elongation activity mediated by the COOH-terminal TPP1-interacting domain was telomere protective by itself. Importantly, a COOH-terminally truncated variant (v5), which consists of the NH 2 -terminal OB folds and the central region of unknown function, was found to protect telomeres and prevent cellular senescence as efficiently as v1. Our data revealed mechanistic and functional differences between v1 and v5: (a) v1, but not v5, functions through the maintenance of telomeric 3 ¶ overhangs; (b) p53 is indispensable to v5 knockdown-induced senescence; and (c) v5 functions at only a fraction of telomeres to prevent DNA damage signaling. Furthermore, v5 was preferentially expressed in mismatch repair (MMR)-deficient cells and tumor tissues, suggesting its role in chromosome stability associated with MMR deficiency. This study highlights a human-specific complexity in telomere protection and damage signaling conferred by functionally distinct isoforms from the single

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Section: Cancer Researchmentioning

confidence: 99%

Functional Diversity of Human Protection of Telomeres 1 Isoforms in Telomere Protection and Cellular Senescence

et al. 2007

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“…At physiologically relevant ionic conditions (∼150 mM K + ), GQs are thermodynamically more stable than competing Watson-Crick pairing (15). These structures were often regarded as obstacles for recruitment of telomerase (16) and translocation of the DNA replication machinery (17), and their unfolding requires helicase activity (17)(18)(19) or ssDNA binding proteins (20,21). It remains unclear whether GQ formation of ssTEL plays any role in protection of telomeres.…”

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confidence: 99%

G-quadruplex formation in telomeres enhances POT1/TPP1 protection against RPA binding

Ray

Bandaria

Qureshi

et al. 2014

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

142

144

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Human telomeres terminate with a single-stranded 3′ G overhang, which can be recognized as a DNA damage site by replication protein A (RPA). The protection of telomeres (POT1)/POT1-interacting protein 1 (TPP1) heterodimer binds specifically to single-stranded telomeric DNA (ssTEL) and protects G overhangs against RPA binding. The G overhang spontaneously folds into various G-quadruplex (GQ) conformations. It remains unclear whether GQ formation affects the ability of POT1/TPP1 to compete against RPA to access ssTEL. Using single-molecule Förster resonance energy transfer, we showed that POT1 stably loads to a minimal DNA sequence adjacent to a folded GQ. At 150 mM K + , POT1 loading unfolds the antiparallel GQ, as the parallel conformation remains folded. POT1/TPP1 loading blocks RPA's access to both folded and unfolded telomeres by two orders of magnitude. This protection is not observed at 150 mM Na + , in which ssTEL forms only a lessstable antiparallel GQ. These results suggest that GQ formation of telomeric overhangs may contribute to suppression of DNA damage signals.telomere protection | DNA damage response | single molecule imaging H uman telomeres consist of 2,000-30,000 bp of doublestranded TTAGGG repeats and terminate with a 50-to 200-nt-long, single-stranded 3′ G overhang (1). Telomeric termini need to be protected against DNA damage signals to ensure genome integrity. Replication protein A (RPA) nonspecifically binds ssDNA, is highly abundant in eukaryotes, and plays a role in DNA replication and repair (2). RPA binding to ssDNA, including telomeric overhangs, activates the ataxia telangiectasia and Rad3-related checkpoint (2, 3). The protection of telomeres (POT1)/telomere protection protein (TPP1) subunit of the shelterin complex contributes to telomere protection by specifically binding to the G overhang (4, 5). RPA is 1,000-fold more abundant than POT1/TPP1 and has a similar affinity for singlestranded telomeric DNA (ssTEL) (6, 7). Therefore, POT1/TPP1 alone may not be able to effectively compete against RPA binding (8, 9). Efficient protection of ssTEL against RPA binding may require association of POT1/TPP1 to the rest of the shelterin complex along double-stranded telomeric tracts or other telomere-associated proteins (6, 9).Another potentially significant factor that could influence the competition between RPA and POT1/TPP1 is the ability of ssTEL to form G-quadruplex (GQ) structures (10, 11). Recent studies have shown that human telomeres form GQ structures in vivo (12, 13) and in cell extracts (14). At physiologically relevant ionic conditions (∼150 mM K + ), GQs are thermodynamically more stable than competing Watson-Crick pairing (15). These structures were often regarded as obstacles for recruitment of telomerase (16) and translocation of the DNA replication machinery (17), and their unfolding requires helicase activity (17-19) or ssDNA binding proteins (20,21). It remains unclear whether GQ formation of ssTEL plays any role in protection of telomeres. ssTEL sequences fold into parallel...

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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.…”

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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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Human replication protein A unfolds telomeric G-quadruplexes

Cited by 168 publications

References 51 publications

Functional Diversity of Human Protection of Telomeres 1 Isoforms in Telomere Protection and Cellular Senescence

Functional Diversity of Human Protection of Telomeres 1 Isoforms in Telomere Protection and Cellular Senescence

G-quadruplex formation in telomeres enhances POT1/TPP1 protection against RPA binding

Cellular Functions of Human RPA1

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