Cooperative interaction of CST and RECQ4 resolves G-quadruplexes and maintains telomere stability.

Human CST (CTC1-STN1-TEN1) is a ssDNA-binding complex that interacts with the replisome to aid in stalled fork rescue. We previously found that CST promotes telomere replication to maintain genomic integrity via G-quadruplex (G4) resolution. However, the detailed mechanism by which CST resolves G4s in vivo and whether additional factors are involved remains unclear. Here, we identify RECQ4 as a novel CST-interacting partner and show that RECQ4 can unwind G4 structures in vitro using a FRET assay. Moreover, G4s accumulate at the telomere after RECQ4 depletion, resulting in telomere dysfunction, including the formation of MTSs, SFEs, and TIFs, suggesting that RECQ4 is crucial for telomere integrity. Furthermore, CST is also required for RECQ4 telomere or chromatin localization in response to G4 stabilizers. RECQ4 is involved in preserving genomic stability by CST and RECQ4 disruption impairs restart of replication forks stalled by G4s. Overall, our findings highlight the essential roles of CST and RECQ4 in resolving G-rich regions, where they collaborate to resolve G4-induced replication deficiencies and maintain genomic homeostasis.

The DNA-binding protein CST associates with the cohesin complex and promotes chromosome cohesion.

Sister chromatid cohesion (SCC), the pairing of sister chromatids after DNA replication until mitosis, is established by loading of the cohesin complex on newly replicated chromatids. Cohesin must then be maintained until mitosis to prevent segregation defects and aneuploidy. However, how SCC is established and maintained until mitosis remains incompletely understood, and emerging evidence suggests that replication stress may lead to premature SCC loss. Here, we report that the ssDNA-binding protein CTC1-STN1-TEN1 (CST) aids in SCC. CST primarily functions in telomere length regulation but also has known roles in replication restart and DNA repair. After depletion of CST subunits, we observed an increase in the complete loss of SCC. In addition, we determined that CST associates with the cohesin complex. Unexpectedly, we did not find evidence of altered cohesin loading or mitotic progression in the absence of CST; however, we did find that treatment with various replication inhibitors increased the association between CST and cohesin. Because replication stress was recently shown to induce SCC loss, we hypothesized that CST may be required to maintain or remodel SCC after DNA replication fork stalling. In agreement with this idea, SCC loss was greatly increased in CST-depleted cells after exogenous replication stress. Based on our findings, we propose that CST aids in the maintenance of SCC at stalled replication forks to prevent premature cohesion loss.

Human CST promotes telomere duplex replication and general replication restart after fork stalling.

Mammalian CST (CTC1-STN1-TEN1) associates with telomeres and depletion of CTC1 or STN1 causes telomere defects. However, the function of mammalian CST remains poorly understood. We show here that depletion of CST subunits leads to both telomeric and non-telomeric phenotypes associated with DNA replication defects. Stable knockdown of CTC1 or STN1 increases the incidence of anaphase bridges and multi-telomeric signals, indicating genomic and telomeric instability. STN1 knockdown also delays replication through the telomere indicating a role in replication fork passage through this natural barrier. Furthermore, we find that STN1 plays a novel role in genome-wide replication restart after hydroxyurea (HU)-induced replication fork stalling. STN1 depletion leads to reduced EdU incorporation after HU release. However, most forks rapidly resume replication, indicating replisome integrity is largely intact and STN1 depletion has little effect on fork restart. Instead, STN1 depletion leads to a decrease in new origin firing. Our findings suggest that CST rescues stalled replication forks during conditions of replication stress, such as those found at natural replication barriers, likely by facilitating dormant origin firing.

Human CST Stimulates Base Excision Repair to Prevent the Accumulation of Oxidative DNA Damage.

CTC1-STN1-TEN1 (CST) is a single-stranded DNA binding protein vital for telomere length maintenance with additional genome-wide roles in DNA replication and repair. While CST was previously shown to function in double-strand break repair and promote replication restart, it is currently unclear whether it has specialized roles in other DNA repair pathways. Proper and efficient repair of DNA is critical to protecting genome integrity. Telomeres and other G-rich regions are strongly predisposed to oxidative DNA damage in the form of 8-oxoguanines, which are typically repaired by the base-excision repair (BER) pathway. Moreover, recent studies suggest that CST functions in the repair of oxidative DNA lesions. Therefore, we tested whether CST interacts with and regulates BER protein activity. Here, we show that CST robustly stimulates proteins involved in BER, including OGG1, Pol ß, APE1, and LIGI, on both telomeric and non-telomeric DNA substrates. Biochemical reconstitution of the pathway indicates that CST stimulates BER. Finally, knockout of STN1 or CTC1 leads to increased levels of 8-oxoguanine, suggesting defective BER in the absence of CST. Combined, our results define an undiscovered function of CST in BER, where it acts as a stimulatory factor to promote efficient genome-wide oxidative repair.

Progerin Can Induce DNA Damage in the Absence of Global Changes in Replication or Cell Proliferation.

Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare genetic condition characterized by features of accelerated aging, and individuals with HGPS seldom live beyond their mid-teens. The syndrome is commonly caused by a point mutation in the LMNA gene which codes for lamin A and its splice variant lamin C, components of the nuclear lamina. The mutation causing HGPS leads to production of a truncated, farnesylated form of lamin A referred to as "progerin." Progerin is also expressed at low levels in healthy individuals and appears to play a role in normal aging. HGPS is associated with an accumulation of genomic DNA double-strand breaks (DSBs) and alterations in the nature of DSB repair. The source of DSBs in HGPS is often attributed to stalling and subsequent collapse of replication forks in conjunction with faulty recruitment of repair factors to damage sites. In this work, we used a model system involving immortalized human cell lines to investigate progerin-induced genomic damage. Using an immunofluorescence approach to visualize phosphorylated histone H2AX foci which mark sites of genomic damage, we report that cells engineered to express progerin displayed a significant elevation of endogenous damage in the absence of any change in the cell cycle profile or doubling time of cells. Genomic damage was enhanced and persistent in progerin-expressing cells treated with hydroxyurea. Overexpression of wild-type lamin A did not elicit the outcomes associated with progerin expression. Our results show that DNA damage caused by progerin can occur independently from global changes in replication or cell proliferation.

Maintaining the end: roles of telomere proteins in end-protection, telomere replication and length regulation.

Chromosome end protection is essential to protect genome integrity. Telomeres, tracts of repetitive DNA sequence and associated proteins located at the chromosomal terminus, serve to safeguard the ends from degradation and unwanted double strand break repair. Due to the essential nature of telomeres in protecting the genome, a number of unique proteins have evolved to ensure that telomere length and structure are preserved. The inability to properly maintain telomeres can lead to diseases such as dyskeratosis congenita, pulmonary fibrosis and cancer. In this review, we will discuss the known functions of mammalian telomere-associated proteins, their role in telomere replication and length regulation and how these processes relate to genome instability and human disease.

Dna2 is a structure-specific nuclease, with affinity for 5'-flap intermediates.

Dna2 is a nuclease/helicase with proposed roles in DNA replication, double-strand break repair and telomere maintenance. For each role Dna2 is proposed to process DNA substrates with a 5'-flap. To date, however, Dna2 has not revealed a preference for binding or cleavage of flaps over single-stranded DNA. Using DNA binding competition assays we found that Dna2 has substrate structure specificity. The nuclease displayed a strong preference for binding substrates with a 5'-flap or some variations of flap structure. Further analysis revealed that Dna2 recognized and bound both the single-stranded flap and portions of the duplex region immediately downstream of the flap. A model is proposed in which Dna2 first binds to a flap base, and then the flap threads through the protein with periodic cleavage, to a terminal flap length of approximately 5 nt. This resembles the mechanism of flap endonuclease 1, consistent with cooperation of these two proteins in flap processing.

Cis- and trans-resveratrol have opposite effects on histone serine-ADP-ribosylation and tyrosine induced neurodegeneration.

Serum tyrosine levels increase during aging, neurocognitive, metabolic, and cardiovascular disorders. However, calorie restriction (CR) and sleep lower serum tyrosine levels. We previously showed that tyrosine inhibits tyrosyl-tRNA synthetase (TyrRS)-mediated activation of poly-ADP-ribose polymerase 1 (PARP1). Here, we show that histone serine-ADP-ribosylation is decreased in Alzheimer's Disease (AD) brains, and increased tyrosine levels deplete TyrRS and cause neuronal DNA damage. However, dopamine and brain-derived neurotrophic factor (BDNF) increase TyrRS and histone serine-ADP-ribosylation. Furthermore, cis-resveratrol (cis-RSV) that binds to TyrRS mimicking a 'tyrosine-free' conformation increases TyrRS, facilitates histone serine-ADP-ribosylation-dependent DNA repair, and provides neuroprotection in a TyrRS-dependent manner. Conversely, trans-RSV that binds to TyrRS mimicking a 'tyrosine-like' conformation decreases TyrRS, inhibits serine-ADP-ribosylation-dependent DNA repair, and induces neurodegeneration in rat cortical neurons. Our findings suggest that age-associated increase in serum tyrosine levels may effect neurocognitive and metabolic disorders and offer a plausible explanation for divergent results obtained in clinical trials using resveratrol.

To Join or Not to Join: Decision Points Along the Pathway to Double-Strand Break Repair vs. Chromosome End Protection.

The regulation of DNA double-strand breaks (DSBs) and telomeres are diametrically opposed in the cell. DSBs are considered one of the most deleterious forms of DNA damage and must be quickly recognized and repaired. Telomeres, on the other hand, are specialized, stable DNA ends that must be protected from recognition as DSBs to inhibit unwanted chromosome fusions. Decisions to join DNA ends, or not, are therefore critical to genome stability. Yet, the processing of telomeres and DSBs share many commonalities. Accordingly, key decision points are used to shift DNA ends toward DSB repair vs. end protection. Additionally, DSBs can be repaired by two major pathways, namely homologous recombination (HR) and non-homologous end joining (NHEJ). The choice of which repair pathway is employed is also dictated by a series of decision points that shift the break toward HR or NHEJ. In this review, we will focus on these decision points and the mechanisms that dictate end protection vs. DSB repair and DSB repair choice.

Human CTC1 promotes TopBP1 stability and CHK1 phosphorylation in response to telomere dysfunction and global replication stress.

CST (CTC1-STN1-TEN1) is a heterotrimeric, RPA-like complex that binds to single-stranded DNA (ssDNA) and functions in the replication of telomeric and non-telomeric DNA. Previous studies demonstrated that deletion of CTC1 results in decreased cell proliferation and telomere DNA damage signaling. However, a detailed analysis of the consequences of conditional CTC1 knockout (KO) has not been fully elucidated. Here, we investigated the effects of CTC1 KO on cell cycle progression, genome-wide replication and activation of the DNA damage response. Consistent with previous findings, we demonstrate that CTC1 KO results in decreased cell proliferation, G2 arrest and RPA-bound telomeric ssDNA. However, despite the increased levels of telomeric RPA-ssDNA, global ATR-dependent CHK1 and p53 phosphorylation was not detected in CTC1 KO cells. Nevertheless, we show that RPA-ssDNA does activate ATR, leading to the phosphorylation of RPA and autophosphorylation of ATR. Further analysis determined that inactivation of ATR, but not CHK1 or ATM, suppressed the accumulation of G2 arrested cells and phosphorylated RPA following CTC1 removal. These results suggest that ATR is localized and active at telomeres but is unable to elicit a global checkpoint response through CHK1. Furthermore, CTC1 KO inhibited CHK1 phosphorylation following hydroxyurea-induced replication stress. Additional studies revealed that this suppression of CHK1 phosphorylation, following replication stress, is caused by decreased levels of the ATR activator TopBP1. Overall, our results identify CST as a novel regulator of the ATR-CHK1 pathway.

FISHing for Damage on Metaphase Chromosomes.

Fluorescence in situ hybridization (FISH) is used to examine chromosomal abnormalities and DNA damage. Developed in the early 1980s, this technique remains an important tool for understanding chromosome biology and diagnosing genetic disease and cancer. Use of FISH on metaphase chromosomes allows the visualization of chromosomal abnormalities at specific loci. Here, we describe methods for creating metaphase chromosome spreads and the use of telomere FISH probes to detect chromosome ends.

Human CST suppresses origin licensing and promotes AND-1/Ctf4 chromatin association.

Human CTC1-STN1-TEN1 (CST) is an RPA-like single-stranded DNA-binding protein that interacts with DNA polymerase α-primase (pol α) and functions in telomere replication. Previous studies suggest that CST also promotes replication restart after fork stalling. However, the precise role of CST in genome-wide replication remains unclear. In this study, we sought to understand whether CST alters origin licensing and activation. Replication origins are licensed by loading of the minichromosome maintenance 2-7 (MCM) complex in G1 followed by replisome assembly and origin firing in S-phase. We find that CST directly interacts with the MCM complex and disrupts binding of CDT1 to MCM, leading to decreased origin licensing. We also show that CST enhances replisome assembly by promoting AND-1/pol α chromatin association. Moreover, these interactions are not dependent on exogenous replication stress, suggesting that CST acts as a specialized replication factor during normal replication. Overall, our findings implicate CST as a novel regulator of origin licensing and replisome assembly/fork progression through interactions with MCM, AND-1, and pol α.

Emerging roles of CST in maintaining genome stability and human disease.

The human CTC1-STN1-TEN1 (CST) complex is a single-stranded DNA binding protein that shares homology with RPA and interacts with DNA polymerase alpha/primase. CST complexes are conserved from yeasts to humans and function in telomere maintenance. A common role of CST across species is in the regulation of telomere extension by telomerase and C-strand fill-in synthesis. However, recent studies also indicate that CST promotes telomere duplex replication as well the rescue of stalled DNA replication at non-telomeric sites. Furthermore, CST dysfunction and mutation is associated with several genetic diseases and cancers. In this review, we will summarize what is known about CST with a particular focus on the emerging roles of CST in DNA replication and human disease.

Human CST has independent functions during telomere duplex replication and C-strand fill-in.

Human CST (CTC1-STN1-TEN1) is an RPA-like complex that is needed for efficient replication through the telomere duplex and genome-wide replication restart after fork stalling. Here, we show that STN1/CST has a second function in telomere replication during G-overhang maturation. Analysis of overhang structure after STN1 depletion revealed normal kinetics for telomerase-mediated extension in S phase but a delay in subsequent overhang shortening. This delay resulted from a defect in C-strand fill-in. Short telomeres exhibited the fill-in defect but normal telomere duplex replication, indicating that STN1/CST functions independently in these processes. Our work also indicates that the requirement for STN1/CST in telomere duplex replication correlates with increasing telomere length and replication stress. Our results provide direct evidence that STN1/CST participates in C-strand fill-in. They also demonstrate that STN1/CST participates in two mechanistically separate steps during telomere replication and identify CST as a replication factor that solves diverse replication-associated problems.

Evolution of CST function in telomere maintenance.

Telomeres consist of an elaborate, higher-order DNA architecture, and a suite of proteins that provide protection for the chromosome terminus by blocking inappropriate recombination and nucleolytic attack. In addition, telomeres facilitate telomeric DNA replication by physical interactions with telomerase and the lagging strand replication machinery. The prevailing view has been that two distinct telomere capping complexes evolved, shelterin in vertebrates and a trimeric complex comprised of Cdc13, Stn1 and Ten1 (CST) in yeast. The recent discovery of a CST-like complex in plants and humans raises new questions about the composition of telomeres and their regulatory mechanisms in multicellular eukaryotes. In this review we discuss the evolving functions and interactions of CST components and their contributions to chromosome end protection and DNA replication.

Significance of the dissociation of Dna2 by flap endonuclease 1 to Okazaki fragment processing in Saccharomyces cerevisiae.

Okazaki fragments are initiated by short RNA/DNA primers, which are displaced into flap intermediates for processing. Flap endonuclease 1 (FEN1) and Dna2 are responsible for flap cleavage. Replication protein A (RPA)-bound flaps inhibit cleavage by FEN1 but stimulate Dna2, requiring that Dna2 cleaves prior to FEN1. Upon cleavage, Dna2 leaves a short flap, which is then cut by FEN1 forming a nick for ligation. Both enzymes require a flap with a free 5'-end for tracking to the cleavage sites. Previously, we demonstrated that FEN1 disengages the tracking mechanism of Dna2 to remove it from the flap. To determine why the disengagement mechanism evolved, we measured FEN1 dissociation of Dna2 on short RNA and DNA flaps, which occur during flap processing. Dna2 tracked onto these flaps but could not cleave, presenting a block to FEN1 entry. However, FEN1 disengaged these nonproductively bound Dna2 molecules, proceeding on to conduct proper cleavage. These results clarify the importance of disengagement. Additional results showed that flap substrate recognition and tracking by FEN1, as occur during fragment processing, are required for effective displacement of the flap-bound Dna2. Dna2 was recently shown to dissociate flap-bound RPA, independent of cleavage. Using a nuclease-defective Dna2 mutant, we reconstituted the sequential dissociation reactions in the proposed RPA/Dna2/FEN1 pathway showing that, even without cutting, Dna2 enables FEN1 to cleave RPA-coated flaps. In summary, RPA, Dna2, and FEN1 have evolved highly coordinated binding properties enabling one protein to succeed the next for proper and efficient Okazaki flap processing.

Dynamic removal of replication protein A by Dna2 facilitates primer cleavage during Okazaki fragment processing in Saccharomyces cerevisiae.

Eukaryotic Okazaki fragments are initiated by a RNA/DNA primer, which is removed before the fragments are joined. Polymerase delta 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.

Flap endonuclease disengages Dna2 helicase/nuclease from Okazaki fragment flaps.

Okazaki fragments contain an initiator RNA/DNA primer that must be removed before the fragments are joined. In eukaryotes, the primer region is raised into a flap by the strand displacement activity of DNA polymerase delta. The Dna2 helicase/nuclease and then flap endonuclease 1 (FEN1) are proposed to act sequentially in flap removal. Dna2 and FEN1 both employ a tracking mechanism to enter the flap 5' end and move toward the base for cleavage. In the current model, Dna2 must enter first, but FEN1 makes the final cut at the flap base, raising the issue of how FEN1 passes the Dna2. To address this, nuclease-inactive Dna2 was incubated with a DNA flap substrate and found to bind with high affinity. FEN1 was then added, and surprisingly, there was little inhibition of FEN1 cleavage activity. FEN1 was later shown, by gel shift analysis, to remove the wild type Dna2 from the flap. RNA can be cleaved by FEN1 but not by Dna2. Pre-bound wild type Dna2 was shown to bind an RNA flap but not inhibit subsequent FEN1 cleavage. These results indicate that there is a novel interaction between the two proteins in which FEN1 disengages the Dna2 tracking mechanism. This interaction is consistent with the idea that the two proteins have evolved a special ability to cooperate in Okazaki fragment processing.

Can the DCoHalpha isozyme compensate in patients with 4a-hydroxy-tetrahydrobiopterin dehydratase/DCoH deficiency?

4a-Hydroxy-tetrahydrobiopterin dehydratase/DCoH is a bifunctional protein. In the cytoplasm it is an enzyme required for the regeneration of tetrahydrobiopterin, an essential cofactor for phenylalanine hydroxylase. In the nucleus it functions as a transcriptional coactivator by forming a 2:2 heterotetramer with the hepatic nuclear factor HNF1alpha (HNF1). Patients with a deficiency of dehydratase activity have elevated levels of phenylalanine, and accumulate 7-pterins due to degradation of its substrate 4a-hydroxy-tetrahydrobiopterin. Curiously, the hyperphenylalaninemia is transient, and no defects in the transcriptional coactivator function have been reported. Recently, a human isozyme, dehydratase/DCoHalpha, has been detected which shares 60% identity with dehydratase/DCoH. This investigation was undertaken to ascertain if dehydratase/DCoHalpha has the pre-requisite properties to compensate in individuals lacking an active form of DCoH. DCoHalpha demonstrated the ability to quantitatively alter HNF1-dependent DNA-binding in vitro whereas DCoH was ineffective in vitro. This characteristic, due to the presence of dimeric DCoHalpha, demonstrates that DCoHalpha does not require any additional mammalian regulation process to alter DNA binding and therefore, may be more effective than DCoH at low concentrations. The dehydratase activity of each isoform was measured by a direct spectrophotometric assay. Km and Vmax for DCoHalpha were both 2-3 times higher than for DCoH, thus leaving the catalytic efficiency (Vmax/Km) the same for both enzymes. In conclusion, the properties of dehydratase/DCoHalpha are consistent with the hypothesis that the activity of this isozyme could account for the relatively mild symptoms reported for patients with a defect in dehydratase/DCoH.