Heterozygous RPA2 variant as a novel genetic cause of telomere biology disorders.

Premature telomere shortening or telomere instability is associated with a group of rare and heterogeneous diseases collectively known as telomere biology disorders (TBDs). Here we identified two unrelated individuals with clinical manifestations of TBDs and short telomeres associated with the identical monoallelic variant c.767A>G; Y256C in RPA2 Although the replication protein A2 (RPA2) mutant did not affect ssDNA binding and G-quadruplex-unfolding properties of RPA, the mutation reduced the affinity of RPA2 with the ubiquitin ligase RFWD3 and reduced RPA ubiquitination. Using engineered knock-in cell lines, we found an accumulation of RPA at telomeres that did not trigger ATR activation but caused short and dysfunctional telomeres. Finally, both patients acquired, in a subset of blood cells, somatic genetic rescue events in either POT1 genes or TERT promoters known to counteract the accelerated telomere shortening. Collectively, our study indicates that variants in RPA2 represent a novel genetic cause of TBDs. Our results further support the fundamental role of the RPA complex in regulating telomere length and stability in humans.

Oligonucleotide-Based Photoaffinity Probes: Chemical Tools and Applications for Protein Labeling.

A variety of proteins interact with DNA and RNA, including polymerases, histones, ribosomes, transcription factors, and repair enzymes. However, the transient non-covalent nature of these interactions poses challenges for analysis. Introducing a covalent bond between proteins and DNA via photochemical activation of a photosensitive functional group introduced onto nucleic acids offers a means to stabilize these often weak interactions without significantly altering the binding interface. Consequently, photoactivatable oligonucleotides are powerful tools for investigating nucleic acid-protein interactions involved in numerous biological and pathological processes. In this review, we provide a comprehensive overview of the chemical tools developed so far and the different strategies used for incorporating the most commonly used photoreactive reagents into oligonucleotide probes or nucleic acids. Furthermore, we illustrate their application with several examples including protein binding site mapping, identification of protein binding partners, and in cell studies.

Gain-of-function mutations in RPA1 cause a syndrome with short telomeres and somatic genetic rescue.

Human telomere biology disorders (TBD)/short telomere syndromes (STS) are heterogeneous disorders caused by inherited loss-of-function mutations in telomere-associated genes. Here, we identify 3 germline heterozygous missense variants in the RPA1 gene in 4 unrelated probands presenting with short telomeres and varying clinical features of TBD/STS, including bone marrow failure, myelodysplastic syndrome, T- and B-cell lymphopenia, pulmonary fibrosis, or skin manifestations. All variants cluster to DNA-binding domain A of RPA1 protein. RPA1 is a single-strand DNA-binding protein required for DNA replication and repair and involved in telomere maintenance. We showed that RPA1E240K and RPA1V227A proteins exhibit increased binding to single-strand and telomeric DNA, implying a gain in DNA-binding function, whereas RPA1T270A has binding properties similar to wild-type protein. To study the mutational effect in a cellular system, CRISPR/Cas9 was used to knock-in the RPA1E240K mutation into healthy inducible pluripotent stem cells. This resulted in severe telomere shortening and impaired hematopoietic differentiation. Furthermore, in patients with RPA1E240K, we discovered somatic genetic rescue in hematopoietic cells due to an acquired truncating cis RPA1 mutation or a uniparental isodisomy 17p with loss of mutant allele, coinciding with stabilized blood counts. Using single-cell sequencing, the 2 somatic genetic rescue events were proven to be independently acquired in hematopoietic stem cells. In summary, we describe the first human disease caused by germline RPA1 variants in individuals with TBD/STS.

GGGCTA repeats can fold into hairpins poorly unfolded by replication protein A: a possible origin of the length-dependent instability of GGGCTA variant repeats in human telomeres.

Human telomeres are composed of GGGTTA repeats and interspersed with variant repeats. The GGGCTA variant motif was identified in the proximal regions of human telomeres about 10 years ago and was shown to display a length-dependent instability. In parallel, a structural study showed that four GGGCTA repeats folded into a non-canonical G-quadruplex (G4) comprising a Watson-Crick GCGC tetrad. It was proposed that this non-canonical G4 might be an additional obstacle for telomere replication. In the present study, we demonstrate that longer GGGCTA arrays fold into G4 and into hairpins. We also demonstrate that replication protein A (RPA) efficiently binds to GGGCTA repeats structured into G4 but poorly binds to GGGCTA repeats structured into hairpins. Our results (along with results obtained with a more stable variant motif) suggest that GGGCTA hairpins are at the origin of GGGCTA length-dependent instability. They also suggest, as working hypothesis, that failure of efficient binding of RPA to GGGCTA structured into hairpins might be involved in the mechanism of GGGCTA array instability. On the basis of our present and past studies about telomeric G4 and their interaction with RPA, we propose an original point of view about telomeric G4 and the evolution of telomeric motifs.

Multiple hPOT1-TPP1 cooperatively unfold contiguous telomeric G-quadruplexes proceeding from 3' toward 5', a feature due to a 3'-end binding preference and to structuring of telomeric DNA.

Telomeres are DNA repeated sequences that associate with shelterin proteins and protect the ends of eukaryotic chromosomes. Human telomeres are composed of 5'TTAGGG repeats and ends with a 3' single-stranded tail, called G-overhang, that can be specifically bound by the shelterin protein hPOT1 (human Protection of Telomeres 1). In vitro studies have shown that the telomeric G-strand can fold into stable contiguous G-quadruplexes (G4). In the present study we investigated how hPOT1, in complex with its shelterin partner TPP1, binds to telomeric sequences structured into contiguous G4 in potassium solutions. We observed that binding of multiple hPOT1-TPP1 preferentially proceeds from 3' toward 5'. We explain this directionality in terms of two factors: (i) the preference of hPOT1-TPP1 for the binding site situated at the 3' end of a telomeric sequence and (ii) the cooperative binding displayed by hPOT1-TPP1 in potassium. By comparing binding in K+ and in Li+, we demonstrate that this cooperative behaviour does not stem from protein-protein interactions, but from structuring of the telomeric DNA substrate into contiguous G4 in potassium. Our study suggests that POT1-TPP1, in physiological conditions, might preferentially cover the telomeric G-overhang starting from the 3'-end and proceeding toward 5'.

RPA prevents G-rich structure formation at lagging-strand telomeres to allow maintenance of chromosome ends.

Replication protein A (RPA) is a highly conserved heterotrimeric single-stranded DNA-binding protein involved in DNA replication, recombination, and repair. In fission yeast, the Rpa1-D223Y mutation provokes telomere shortening. Here, we show that this mutation impairs lagging-strand telomere replication and leads to the accumulation of secondary structures and recruitment of the homologous recombination factor Rad52. The presence of these secondary DNA structures correlates with reduced association of shelterin subunits Pot1 and Ccq1 at telomeres. Strikingly, heterologous expression of the budding yeast Pif1 known to efficiently unwind G-quadruplex rescues all the telomeric defects of the D223Y cells. Furthermore, in vitro data show that the identical D to Y mutation in human RPA specifically affects its ability to bind G-quadruplex. We propose that RPA prevents the formation of G-quadruplex structures at lagging-strand telomeres to promote shelterin association and facilitate telomerase action at telomeres.

G4 motifs affect origin positioning and efficiency in two vertebrate replicators.

DNA replication ensures the accurate duplication of the genome at each cell cycle. It begins at specific sites called replication origins. Genome-wide studies in vertebrates have recently identified a consensus G-rich motif potentially able to form G-quadruplexes (G4) in most replication origins. However, there is no experimental evidence to demonstrate that G4 are actually required for replication initiation. We show here, with two model origins, that G4 motifs are required for replication initiation. Two G4 motifs cooperate in one of our model origins. The other contains only one critical G4, and its orientation determines the precise position of the replication start site. Point mutations affecting the stability of this G4 in vitro also impair origin function. Finally, this G4 is not sufficient for origin activity and must cooperate with a 200-bp cis-regulatory element. In conclusion, our study strongly supports the predicted essential role of G4 in replication initiation.

The exception that confirms the rule: a higher-order telomeric G-quadruplex structure more stable in sodium than in potassium.

DNA and RNA guanine-quadruplexes (G4s) are stabilized by several cations, in particular by potassium and sodium ions. Generally, potassium stabilizes guanine-quartet assemblies to a larger extent than sodium; in this article we report about a higher-order G4 structure more stable in sodium than in potassium. Repeats of the DNA GGGTTA telomeric motif fold into contiguous G4 units. Using three independent approaches (thermal denaturation experiments, isothermal molecular-beacon and protein-binding assays), we show that the (GGGTTA)7GGG sequence, folding into two contiguous G4 units, exhibits an unusual feature among G4 motifs: despite a lower thermal stability, its sodium conformation is more stable than its potassium counterpart at physiological temperature. Using differential scanning calorimetry and mutated sequences, we show that this switch in the relative stability of the sodium and potassium conformations (occurring around 45 °C in 100 mM cation concentration) is the result of a more favorable enthalpy change upon folding in sodium, generated by stabilizing interactions between the two G4 units in the sodium conformation. Our work demonstrates that interactions between G4 structural domains can make a higher-order structure more stable in sodium than in potassium, even though its G4 structural domains are individually more stable in potassium than in sodium.

5' to 3' Unfolding Directionality of DNA Secondary Structures by Replication Protein A: G-QUADRUPLEXES AND DUPLEXES.

The replication protein A (RPA) is a single-stranded DNA-binding protein that plays an essential role in DNA metabolism. RPA is able to unfold G-quadruplex (G4) structures formed by telomeric DNA sequences, a function important for telomere maintenance. To elucidate the mechanism through which RPA unfolds telomeric G4s, we studied its interaction with oligonucleotides that adopt a G4 structure extended with a single-stranded tail on either side of the G4. Binding and unfolding was characterized using several biochemical and biophysical approaches and in the presence of specific G4 ligands, such as telomestatin and 360A. Our data show that RPA can bind on each side of the G4 but it unwinds the G4 only from 5' toward 3'. We explain the 5' to 3' unfolding directionality in terms of the 5' to 3' oriented laying out of hRPA subunits along single-stranded DNA. Furthermore, we demonstrate by kinetics experiments that RPA proceeds with the same directionality for duplex unfolding.

Human POT1 is required for efficient telomere C-rich strand replication in the absence of WRN.

Mechanisms of telomere replication remain poorly defined. It has been suggested that G-rich telomeric strand replication by lagging mechanisms requires, in a stochastic way, the WRN protein. Here we show that this requirement is more systematic than previously thought. Our data are compatible with a situation in which, in the absence of WRN, DNA synthesis at replication forks is uncoupled, thus allowing replication to continue on the C strand, while single G strands accumulate. We also show that in cells in which both WRN and POT1 are limiting, both G- and C-rich telomeric strands shorten, suggesting a complete replication block. Under this particular condition, expression of a fragment spanning the two POT1-OB (oligonucleotide-binding) fold domains is able to restore C (but not G) strand replication, suggesting that binding of POT1 to the lagging strand allows DNA synthesis uncoupling in the absence of WRN. Furthermore, in vitro experiments indicate that purified POT1 has a higher affinity for the telomeric G-rich strand than purified RPA. We propose a model in which the relative enrichments of POT1 versus RPA on the telomeric lagging strand allows or does not allow uncoupling of DNA synthesis at the replication fork. Our study reveals an unanticipated role for hPOT1 during telomere replication.

Evidence for direct contact between the RPA3 subunit of the human replication protein A and single-stranded DNA.

Replication Protein A is a single-stranded (ss) DNA-binding protein that is highly conserved in eukaryotes and plays essential roles in many aspects of nucleic acid metabolism, including replication, recombination, DNA repair and telomere maintenance. It is a heterotrimeric complex consisting of three subunits: RPA1, RPA2 and RPA3. It possesses four DNA-binding domains (DBD), DBD-A, DBD-B and DBD-C in RPA1 and DBD-D in RPA2, and it binds ssDNA via a multistep pathway. Unlike the RPA1 and RPA2 subunits, no ssDNA-RPA3 interaction has as yet been observed although RPA3 contains a structural motif found in the other DBDs. We show here using 4-thiothymine residues as photoaffinity probe that RPA3 interacts directly with ssDNA on the 3'-side on a 31 nt ssDNA.

The meiosis-specific MEIOB-SPATA22 complex cooperates with RPA to form a compacted mixed MEIOB/SPATA22/RPA/ssDNA complex.

During meiosis, programmed double-strand breaks are repaired by homologous recombination (HR) to form crossovers that are essential to homologous chromosome segregation. Single-stranded DNA (ssDNA) containing intermediates are key features of HR, which must be highly regulated. RPA, the ubiquitous ssDNA binding complex, was thought to play similar roles during mitotic and meiotic HR until the recent discovery of MEIOB and its partner, SPATA22, two essential meiosis-specific proteins. Here, we show that like MEIOB, SPATA22 resembles RPA subunits and binds ssDNA. We studied the physical and functional interactions existing between MEIOB, SPATA22, and RPA, and show that MEIOB and SPATA22 interact with the preformed RPA complex through their interacting domain and condense RPA-coated ssDNA in vitro. In meiotic cells, we show that MEIOB and SPATA22 modify the immunodetection of the two large subunits of RPA. Given these results, we propose that MEIOB-SPATA22 and RPA form a functional ssDNA-interacting complex to satisfy meiotic HR requirements by providing specific properties to the ssDNA.

Functional Diversification of Replication Protein A Paralogs and Telomere Length Maintenance in Arabidopsis.

Replication protein A (RPA) is essential for many facets of DNA metabolism. The RPA gene family expanded in Arabidopsis thaliana with five phylogenetically distinct RPA1 subunits (RPA1A-E), two RPA2 (RPA2A and B), and two RPA3 (RPA3A and B). RPA1 paralogs exhibit partial redundancy and functional specialization in DNA replication (RPA1B and RPA1D), repair (RPA1C and RPA1E), and meiotic recombination (RPA1A and RPA1C). Here, we show that RPA subunits also differentially impact telomere length set point. Loss of RPA1 resets bulk telomeres at a shorter length, with a functional hierarchy for replication group over repair and meiosis group RPA1 subunits. Plants lacking RPA2A, but not RPA2B, harbor short telomeres similar to the replication group. Telomere shortening does not correlate with decreased telomerase activity or deprotection of chromosome ends in rpa mutants. However, in vitro assays show that RPA1B2A3B unfolds telomeric G-quadruplexes known to inhibit replications fork progression. We also found that ATR deficiency can partially rescue short telomeres in rpa2a mutants, although plants exhibit defects in growth and development. Unexpectedly, the telomere shortening phenotype of rpa2a mutants is completely abolished in plants lacking the RTEL1 helicase. RTEL1 has been implicated in a variety of nucleic acid transactions, including suppression of homologous recombination. Thus, the lack of telomere shortening in rpa2a mutants upon RTEL1 deletion suggests that telomere replication defects incurred by loss of RPA may be bypassed by homologous recombination. Taken together, these findings provide new insight into how RPA cooperates with replication and recombination machinery to sustain telomeric DNA.

A multi-approach analysis highlights the relevance of RPA-1 as a telomere end-binding protein (TEBP) in Leishmania amazonensis.

BACKGROUND: Telomeres are chromosome end structures important in the maintenance of genome homeostasis. They are replenished by the action of telomerase and associated proteins, such as the OB (oligonucleotide/oligosaccharide-binding)-fold containing telomere-end binding proteins (TEBP) which plays an essential role in telomere maintenance and protection. The nature of TEBPs is well known in higher and some primitive eukaryotes, but it remains undetermined in trypanosomatids. Previous in silico searches have shown that there are no homologs of the classical TEPBs in trypanosomatids, including Leishmania sp. However, Replication Protein A subunit 1 (RPA-1), an OB-fold containing DNA-binding protein, was found co-localized with trypanosomatids telomeres and showed a high preference for the telomeric G-rich strand. METHODS AND RESULTS: We predicted the absence of structural homologs of OB-fold containing TEBPs in the Leishmania sp. genome using structural comparisons. We demonstrated by molecular docking that the ssDNA binding mode of LaRPA-1 shares features with the higher eukaryotes POT1 and RPA-1 crystal structures ssDNA binding mode. Using fluorescence spectroscopy, protein-DNA interaction assays, and FRET, we respectively show that LaRPA-1 shares some telomeric functions with the classical TEBPs since it can bind at least one telomeric repeat, protect the telomeric G-rich DNA from 3'-5' Exonuclease I digestion, and unfold telomeric G-quadruplex. CONCLUSIONS: Our results suggest that RPA-1 emerges as a TEBP in trypanosomatids, and in this context, we present two possible evolutionary landscapes of trypanosomatids RPA-1 that could reflect upon the evolution of OB-fold containing TEBPs from all eukaryotes.

Human replication protein A unfolds telomeric G-quadruplexes.

G-quadruplex structures inhibit telomerase activity and must be disrupted for telomere elongation during S phase. It has been suggested that the replication protein A (RPA) could unwind and maintain single-stranded DNA in a state amenable to the binding of telomeric components. We show here that under near-physiological in vitro conditions, human RPA is able to bind and unfold G-quadruplex structures formed from a 21mer human telomeric sequence. Analyses by native gel electrophoresis, cross-linking and fluorescence resonance energy transfer indicate the formation of both 1:1 and 2:1 complexes in which G-quadruplexes are unfolded. In addition, quadruplex opening by hRPA is much faster than observed with the complementary DNA, demonstrating that this protein efficiently unfolds G-quartets. A two-step mechanism accounting for the binding of hRPA to G-quadruplexes is proposed. These data point to the involvement of hRPA in regulation of telomere maintenance.

Probing hyper-negatively supercoiled mini-circles with nucleases and DNA binding proteins.

It is well accepted that the introduction of negative supercoils locally unwinds the DNA double helix, influencing thus the activity of proteins. Despite the use of recent methods of molecular dynamics simulations to model the DNA supercoiling-induced DNA deformation, the precise extent and location of unpaired bases induced by the negative supercoiling have never been investigated at the nucleotide level. Our goals in this study were to use radiolabeled double-stranded DNA mini-circles (dsMCs) to locate the unpaired bases on dsMCs whose topology ranged from relaxed to hyper-negatively supercoiled states, and to characterize the binding of proteins involved in the DNA metabolism. Our results show that the Nuclease SI is nearly ten times more active on hyper-negatively supercoiled than relaxed DNA. The structural changes responsible for this stimulation of activity were mapped for the first time with a base pair resolution and shown to be subtle and distributed along the entire sequence. As divalent cations modify the DNA topology, our binding studies were conducted with or without magnesium. Without magnesium, the dsMCs topoisomers mostly differ by their twist. Under these conditions, the Escherichia coli topoisomerase I weakly binds relaxed dsMCs and exhibits a stronger binding on negatively and hyper-negatively supercoiled dsMCs than relaxed dsMCs, with no significant difference in the binding activity among the supercoiled topoisomers. For the human replication protein A (hRPA), the more negatively supercoiled is the DNA, the better the binding, illustrating the twist-dependent binding activity for this protein. The presence of magnesium permits the dsMCs to writhe upon introduction of negative supercoiling and greatly modifies the binding properties of the hRPA and Escherichia coli SSB on dsMCs, indicating a magnesium-dependent DNA binding behavior. Finally, our experiments that probe the topology of the DNA in the hRPA-dsMC complexes show that naked and hRPA-bound dsMCs have the same topology.

The binding efficiency of RPA to telomeric G-strands folded into contiguous G-quadruplexes is independent of the number of G4 units.

Replication protein A (RPA) is a single-stranded DNA binding protein involved in replication and in telomere maintenance. During telomere replication, G-quadruplexes (G4) can accumulate on the lagging strand template and need to be resolved. It has been shown that human RPA is able to unfold a single G4. Nevertheless, the G-strand of human telomeres is prone to fold into higher-order structures formed by contiguous G-quadruplexes. To understand how RPA deals with these structures, we studied its interaction with telomeric G-strands folding into an increasing number of contiguous G4s. The aim of this study was to determine whether the efficiency of binding/unfolding of hRPA to telomeric G-strands depends on the number of G4 units. Our data show that the number n of contiguous G4 units (n ≥ 2) does not affect the efficiency of hRPA to coat transiently exposed single-stranded telomeric G-strands. This feature may be essential in preventing instability due to G4 structures during telomere replication.

Photoactivatable oligonucleotide probes to trap single-stranded DNA binding proteins: Updating the potential of 4-thiothymidine from a comparative study.

Photoaffinity labeling (PAL) in combination with recent developments in mass spectrometry is a powerful tool for studying nucleic acid-protein interactions, enabling crosslinking of both partners through covalent bond formation. Such a strategy requires a preliminary study of the most judicious photoreactive group to crosslink efficiently with the target protein. In this study, we report a survey of three different photoreactive nucleobases (including a guanine functionalized with a benzophenone or a diazirine and the zero-length agent 4-thiothymine) incorporated in 30-mer oligonucleotides (ODN) containing a biotin moiety for selective trapping and enrichment of single-stranded DNA binding proteins (SSB). First, the conditions and efficiency of the photochemical reaction with a purified protein using human replication protein A as the relevant model was studied. Secondly, the ability of the probe as bait to photocrosslink and enrich SSB in cell lysate was addressed. Among the different ODN probes studied, we showed that 4-thiothymine was the most relevant: i) it allows efficient and specific trapping of SSB in whole cell extracts in a similar extent as the widely used diazirine, ii) it features the advantages of a zero-length agent thus retaining the physicochemical properties of the ODN bait; iii) ODN including this photochemical agent are easily accessible. In combination with mass spectrometry, the probes incorporating this nucleobase are powerful tools for PAL strategies and can be added in the toolbox of the traditional photocrosslinkers for studying DNA-protein interactions.

Photochemistry of 4-Thiothymine Derivatives in the Presence of N-9-Substituted-Adenine Derivatives: Formation of N-6-Formamidopyrimidines.

UV irradiation of aqueous solutions containing either 4-thiothymin-1-ylacetic acid (1b) and adenosine (2a), 4-thiothymidine (1a) and adenin-9-ylacetic acid (2b), or 1b and 2b led to 4,5-diamino-6-formamidopyrimidine (N-6-Fapy-Ade) derivatives as observed after irradiation of a mixture of 1a and 2a (J. Am. Chem. Soc. 1996, 118, 8142-8143). These new observations demonstrate that the replacement of one or both nucleoside sugar residues by a carboxymethyl group does not affect the regioselective course of the photochemical reaction. The thermal decomposition of 3a that resulted from irradiation of 1a in the presence of 2a, was examined along with its behavior under mild alkaline conditions. Finally, irradiation of N-3-methyl-4-thiothymidine (6a) in the presence of adenosine gave the N-3-methylcytidine derivative 7.

Binding polarity of RPA to telomeric sequences and influence of G-quadruplex stability.

Replication protein A (RPA) is a single-stranded DNA binding protein that plays an essential role in telomere maintenance. RPA binds to and unfolds G-quadruplex (G4) structures formed in telomeric DNA, thus facilitating lagging strand DNA replication and telomerase activity. To investigate the effect of G4 stability on the interactions with human RPA (hRPA), we used a combination of biochemical and biophysical approaches. Our data revealed an inverse relationship between G4 stability and ability of hRPA to bind to telomeric DNA; notably small G4 ligands that enhance G4 stability strongly impaired G4 unfolding by hRPA. To gain more insight into the mechanism of binding and unfolding of telomeric G4 structures by RPA, we carried out photo-crosslinking experiments to elucidate the spatial arrangement of the RPA subunits along the DNA strands. Our results showed that RPA1 and RPA2 are arranged from 5' to 3' along the unfolded telomeric G4, as already described for unstructured single-stranded DNA, while no contact is possible with RPA3 on this short oligonucleotide. In addition, these data are compatible with a 5' to 3' directionality in G4 unfolding by hRPA.