Nucleoprotein Structures Research Articles

Human adenoviral DNA association with nucleosomes containing histone variant H3.3 during the early phase of infection is not dependent on viral transcription or replication.

Adenovirus (Ad) DNA undergoes dynamic changes in protein association as the virus progresses through its replicative cycle. Within the virion, the Ad DNA associates primarily with the virus-encoded, protamine-like protein VII. During the early phase of infection (∼6 h), the viral DNA showed declining association with VII, suggesting that VII was removed from at least some regions of the viral DNA. Within 6 h, the viral DNA was wrapped into a repeating nucleosome-like array containing the histone variant H3.3. Transcription elongation was not required to strip VII from the viral DNA or for deposition of H3.3. H3.1 did not associate with the viral DNA at any point during infection. During the late phase of infection (i.e., active DNA replication ∼12-24 h), association with H3 was dramatically reduced and the repeating nucleosome-like pattern was no longer evident. Thus, we have uncovered some of the changes in nucleoprotein structure that occur during lytic Ad infection.

The effect of intrauterine growth on leukocyte telomere length at birth

Objective: Telomeres are specialized nucleoprotein structures located at the ends of chromosomes, which play a crucial role in genomic stability. Telomere shortening has been proposed as a biomarker for the onset of age-related diseases. This study aimed to determine whether restricted or increased intrauterine growth affects leukocyte telomere length (LTL) at birth.Materials and methods: One hundred sixty-five (n = 165) full-term neonates participated in the study. Fetuses were classified as intrauterine growth restriction (IUGR, n = 21), large-for-gestational-age (LGA, n = 15), or appropriate-for-gestational-age (AGA, n = 129), based on customized birth-weight standards. Mixed arteriovenous cord blood samples were collected for isolation of leukocyte DNA. The LTL was measured using multiplex monochrome quantitative real-time PCR and telomeric restriction fragments through Southern blot analysis (terminal restriction fragment [TRF]).Results: Despite differences among groups in birth weight, length and head circumference, LTL did not differ among AGA (6.78 ± 0.58), IUGR (10.54 ± 1.80), and LGA (11.95 ± 2.42) neonates (p = .098). Cord blood IGF-1 and IGFBP-3 concentrations were higher in the LGA group. LTL positively correlated with birth length (r = 0.176, p = .032).Conclusions: Intrauterine growth does not seem to affect LTL at birth. Further studies, comprising a larger sample size of IUGR, LGA, and AGA neonates, are required to determine whether growth at birth influences LTL.

Melatonin affects the motility and adhesiveness of invitro capacitated boar spermatozoa via a mechanism that does not depend on intracellular ROS levels.

This work sought to address the effects of melatonin during invitro capacitation (IVC) and progesterone-induced acrosome exocytosis (IVAE) in boar spermatozoa. With this purpose, two different experiments were set. In the first one, IVC and IVAE were induced in the absence or presence of melatonin, which was added either at the start of IVC or upon triggering the IVAE with progesterone. Different parameters were evaluated, including intracellular levels of peroxides and superoxides, free cysteine radicals and distribution of specific lectins. While melatonin neither affected most capacitation-associated parameters nor IVAE, it dramatically decreased sperm motility, with a maximal effect at 5μm. This effect was accompanied by a significant increase in the percentage of agglutinated spermatozoa, which was independent from noticeable changes in the distribution of lectins. Levels of free cysteine radicals were significantly lower in melatonin treatments than in the control after 4h of incubation in capacitating medium. The second experiment evaluated the effects of melatonin on invitro fertilising ability of boar spermatozoa. Spermatozoa previously subjected to IVC in the presence of 1μm melatonin and used for invitro fertilisation exhibited less ability to bind the zona pellucida (ZP) and higher percentages of monospermy. In conclusion, melatonin affects sperm motility and the stability of nucleoprotein structure and also modulates the ability of invitro capacitated boar spermatozoa to bind the oocyte ZP. However, such effects do not seem to be related to either its antioxidant properties or changes in the sperm glycocalix.

The BUB3-BUB1 Complex Promotes Telomere DNA Replication

Telomeres and telomere-binding proteins form complex secondary nucleoprotein structures that are critical for genome integrity but can present serious challenges during telomere DNA replication. It remains unclear how telomere replication stress is resolved during S phase. Here, we show that the BUB3-BUB1 complex, a component in spindle assembly checkpoint, binds to telomeres during Sphase and promotes telomere DNA replication. Loss of the BUB3-BUB1 complex results in telomere replication defects, including fragile and shortened telomeres. We also demonstrate that the telomere-binding ability of BUB3 and kinase activity of BUB1 are indispensable to BUB3-BUB1 function at telomeres. TRF2 targets BUB1-BUB3 to telomeres, and BUB1 can directly phosphorylate TRF1 and promote TRF1 recruitment of BLM helicase to overcome replication stress. Our findings have uncovered previously unknown roles for the BUB3-BUB1 complex in S phase and shed light on how proteins from diverse pathways function coordinately to ensure proper telomere replication and maintenance.

A review of telomere length in sarcopenia and frailty.

Sarcopenia and frailty are associated with several important health-related adverse events, including disability, loss of independence, institutionalization and mortality. Sarcopenia can be considered a biological substrate of frailty, and the prevalence of both these conditions progressively increases with age. Telomeres are nucleoprotein structures located at the end of linear chromosomes and implicated in cellular ageing, shorten with age, and are associated with various age-related diseases. In addition, telomere length (TL) is widely considered a molecular/cellular hallmark of the ageing process. This narrative review summarizes the knowledge about telomeres and analyzes for the first time a possible association of TL with sarcopenia and frailty. The overview provided by the present review suggests that leukocyte TL as single measurement, calculated by quantitative real-time polymerase chain reaction(qRT-PCR), cannot be considered a meaningful biological marker for complex, multidimensional age-related conditions, such as sarcopenia and frailty. Panels of biomarkers, including TL, may provide more accurate assessment and prediction of outcomes in these geriatric syndromes in elderly people.

Telomeres and Telomerase: Molecular Views and Perspectives

Telomere, the nucleoprotein structure at the end of eukaryotic linear chromosomes is indispensable for maintaining the genome stability. Telomeric DNA loss is apparent with each cell division, which marks an endpoint to the indefinite replication of the cell by causing replicative senescence that may lead to the programmed cell death. The loss of telomere is normal in cell division and as such after 20 - 40 divisions, telomere becomes too short to facilitate the capping function. Telomere uncapping or chromosomal free end causes a potential threat to the genomic stability and thus leads to the accumulation of chromosomal abnormalities that have been known to play a role in aging and cancer. Telomerase, the ribonucleoprotein complex, and its accessory proteins are required to maintain the telomere sequence. Telomerase plays a key role in maintaining the length of telomere by adding G-rich repeat sequences. Its activity has been found to be quite high in the gametes, stem cells and most importantly tumor cells. Almost 85% of tumor cells compensate for telomere loss aided by telomerase-associated protein complex and shelter in complex or telosome. However, 5% - 10% of the cells undergo telomerase-independent mechanism. This review presents the molecular view of the telomere and telomerase along with its associated complex structures. It also discusses its contrasting role in causing cellular senescence and promoting tumorigenesis.

Building expanded structures from tetrahedral DNA branching elements, RNA and TMV protein.

By combining both chemical and enzymatic ligation with procedures guiding the self-assembly of nanotubular tobacco mosaic virus (TMV)-like particles (TLPs), novel nucleoprotein structures based on DNA-terminated branching elements, RNA scaffolds and TMV coat protein (CP) are made accessible. Tetrahedral tetrakis(hydroxybiphenyl)adamantane cores with four 5'-phosphorylated dinucleotide arms were coupled to DNA linkers by chemical ligation. The resulting three-dimensional (3D) branching elements were enzymatically ligated to the 3' termini of RNA scaffolds either prior to or after the RNAs' incorporation into TLPs. Thus, architectures with interconnected nanotube domains in two different length classes were generated, each containing 70 CP subunits per 10 nm length. Short TMV origin-of-assembly-containing RNA scaffolds ligated to the DNA allowed the growth of protein-coated 34 nm tubes on the terminal RNA strands in situ. Alternatively, 290 nm pre-fabricated tubes with accessible RNA 3' termini, attained by DNA blocking elements hybridized to the RNAs, were ligated with the branched cores. Both approaches resulted in four-armed nanoobjects, demonstrating a so far unique combination of organic synthesis of branching elements, enzymatic modifications, nucleic acid-based scaffolding and RNA-guided and DNA-controlled assembly of tubular RNA-encapsidating protein domains, yielding a novel class of 3D nucleoprotein architectures with polyvalent protein elements. In the long term, the production route might give rise to supramolecular systems with complex functionalities, installed via the orthogonal coupling of effector molecules to TLP domains.

CTCF driven TERRA transcription facilitates completion of telomere DNA replication

Telomere repeat DNA forms a nucleo-protein structure that can obstruct chromosomal DNA replication, especially under conditions of replication stress. Transcription of telomere repeats can initiate at subtelomeric CTCF-binding sites to generate telomere repeat-encoding RNA (TERRA), but the role of transcription, CTCF, and TERRA in telomere replication is not known. Here, we have used CRISPR/Cas9 gene editing to mutate CTCF-binding sites at the putative start site of TERRA transcripts for a class of subtelomeres. Under replication stress, telomeres lacking CTCF-driven TERRA exhibit sister-telomere loss and upon entry into mitosis, exhibit the formation of ultra-fine anaphase bridges and micronuclei. Importantly, these phenotypes could be rescued by the forced transcription of TERRA independent of CTCF binding. Our findings indicate that subtelomeric CTCF facilitates telomeric DNA replication by promoting TERRA transcription. Our findings also demonstrate that CTCF-driven TERRA transcription acts in cis to facilitate telomere repeat replication and chromosome stability.

Expression of Telomere-Associated Proteins is Interdependent to Stabilize Native Telomere Structure and Telomere Dysfunction by G-Quadruplex Ligand Causes TERRA Upregulation.

Telomere DNA can form specialized nucleoprotein structure with telomere-associated proteins to hide free DNA ends or G-quadruplex structures under certain conditions especially in presence of G-quadruplex ligand. Telomere DNA is transcribed to form non-coding telomere repeat-containing RNA (TERRA) whose biogenesis and function is poorly understood. Our aim was to find the role of telomere-associated proteins and telomere structures in TERRA transcription. We silenced four [two shelterin (TRF1, TRF2) and two non-shelterin (PARP-1, SLX4)] telomere-associated genes using siRNA and verified depletion in protein level. Knocking down of one gene modulated expression of other telomere-associated genes and increased TERRA from 10q, 15q, XpYp and XqYq chromosomes in A549 cells. Telomere was destabilized or damaged by G-quadruplex ligand pyridostatin (PDS) and bleomycin. Telomere dysfunction-induced foci (TIFs) were observed for each case of depletion of proteins, treatment with PDS or bleomycin. TERRA level was elevated by PDS and bleomycin treatment alone or in combination with depletion of telomere-associated proteins.

Histone Variants and Disease.

In eukaryotes, the genome is organized into a complex nucleoprotein structure called chromatin. Despite the simplicity of its monomer, DNA and two copies of four histones, the existence of histone variants opens possibilities of multiple chromatin landscapes and fine-tune regulation of molecular mechanisms for the regulation of gene expression and maintenance of genome stability. However, any defects in these combinations may contribute to disease development and/or progression. Here, I review human histone variants and their chaperones, and discuss how they contribute to pathological conditions.

Interplay Among Constitutes of Ebola Virus: Nucleoprotein, Polymerase L, Viral Proteins

Ebola virus is a highly lethal filovirus, claimed thousands of people in its recent outbreak. Seven viral proteins constitute ebola viral structure, and four of them (nucleoprotein (NP), polymerase L, VP35 and VP30) participate majorly in viral replication and transcription. We have elucidated a conformation change of NP cleft by VP35 NP-binding protein domains through superimposing two experimental NP structure images and discussed the function of this conformation change in the replication and transcription with polymerase complex (L, VP35 and VP30). The important roles of VP30 in viral RNA synthesis have also been discussed. A “tapping” model has been proposed in this paper for a better understanding of the interplay among the four viral proteins (NP, polymerase L, VP35 and VP30). Moreover, we have pinpointed some key residue changes on NP (both NP N- and C-terminal) and L between Reston and Zaire by computational studies. Together, this paper provides a description of interactions among ebola viral proteins (NP, L, VP35, VP30 and VP40) in viral replication and transcription, and sheds light on the complex system of viral reproduction.

Age at maturation has sex- and temperature-specific effects on telomere length in a fish.

Telomeres are highly conserved nucleoprotein structures which protect genome integrity. The length of telomeres is influenced by both genetic and environmental factors, but relatively little is known about how different hereditary and environmental factors interact in determining telomere length. We manipulated growth rates and timing of maturation by exposing full-sib nine-spined sticklebacks (Pungitius pungitius) to two different temperature treatments and quantified the effects of temperature treatments, sex, timing of maturation, growth rate and family (genetic influences) on telomere length. We did not find the overall effect of temperature treatment on the relative telomere length. However, we found that variation in telomere length was related to timing of maturation in a sex- and temperature-dependent manner. Telomere length was negatively related to age at maturation in elevated temperature and early maturing males and females differed in telomere length. Variation in growth rate did not explain any variation in telomere length. The broad sense heritability (h 2) of telomere length was estimated at h 2=0.31-0.47, suggesting predominance of environmental over genetic determinants of telomere length variability. This study provides the first evidence that age at maturation together with factors associated with it are influencing telomere length in an ectotherm. Future studies are encouraged to identify the extent to which these results can be replicated in other ectotherms.

Prognostic Significance of Telomere Length in newly diagnosed Chronic Lymphocytic Leukemia patients: measured By FlowFISH technique

Telomeres are specialized nucleoprotein structures present at the ends of human chromosomes, andare constituted of a tandem of repeats TTAGGG. Telomere length measurement has been assessed by many previousstudies In B-Chronic Lymphocytic Leukemia patients (B-CLL). Telomere shortening was found to correlate withdisease progression. The aim of our study was to measure Telomere Length (TL) by Florescence In-SituHybridization coupled with Flow-cytometer (Flow-FISH) technique in newly diagnosed B-CLL patients. Explore itsprognostic significance, through determination of the relationship between TL; with disease stage, other prognosticmarker such as CD38 & ZAP-70 expression and patient response to treatment.

Abstract 3469: Identification of novel human telomerase associated protein by mass spectrometry

Abstract Telomeres are nucleoprotein structures at the ends of eukaryotic chromosomes that prevent them from degradation, recombination, and end-to-end fusion. Telomere consists of DNA repeats, which in human is of the sequence TTAGGG, and it is bound by a group of proteins called shelterin complex. Telomere is synthesized by telomerase. Telomerase is absent in most human somatic cells while it is up-regulated in 90% of cancer cells, in which telomerase plays an essential role in cancer cells’ replicative immortality. However, telomerase is only present at approximately 250 molecules per cancer cell. To exert its function of elongating telomere, telomerase has to find the end of telomeres which needed to be elongated. However, how telomerase is precisely recruited to telomere ends is not clearly understood. To better understand the recruitment process of telomerase, we initially screened and subsequently identified novel binding proteins of hTERT, the catalytic subunit of telomerase, through a pull-down assay using cancer cell lines with endogenously Flag-tagged hTERT. Using mass spectrometry (MS), we were able to identify a promising interesting protein. Further, direct interaction between TERT and this protein was validated. Then, we found that this protein can also interact with the shelterin component, TRF1 (Telomeric repeat-binding factor 1), which binds to double stranded telomeric DNA. Furthermore, from previous published reports, this protein was shown to interact with single-stranded telomeric DNA binding protein POT1 (Protection of Telomeres 1) in the shelterin complex. Functionally, overexpression of this protein in cancer cell lines can result in the elongation of telomeres, while knock-down or knock-out of the protein results in the shortening of telomere. In conclusion, we identified an hTERT-binding protein that affects telomere maintenance which may be involved in telomerase recruitment process. In the long run, it could serve as a potential therapeutic target for cancers. Citation Format: Jing Wang, Xuezhi Bi, Shang Li. Identification of novel human telomerase associated protein by mass spectrometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3469. doi:10.1158/1538-7445.AM2017-3469

A phosphorylation-and-ubiquitylation circuitry driving ATR activation and homologous recombination

RPA-coated single-stranded DNA (RPA–ssDNA), a nucleoprotein structure induced by DNA damage, promotes ATR activation and homologous recombination (HR). RPA is hyper-phosphorylated and ubiquitylated after DNA damage. The ubiquitylation of RPA by PRP19 and RFWD3 facilitates ATR activation and HR, but how it is stimulated by DNA damage is still unclear. Here, we show that RFWD3 binds RPA constitutively, whereas PRP19 recognizes RPA after DNA damage. The recruitment of PRP19 by RPA depends on PIKK-mediated RPA phosphorylation and a positively charged pocket in PRP19. An RPA32 mutant lacking phosphorylation sites fails to recruit PRP19 and support RPA ubiquitylation. PRP19 mutants unable to bind RPA or lacking ubiquitin ligase activity also fail to support RPA ubiquitylation and HR. These results suggest that RPA phosphorylation enhances the recruitment of PRP19 to RPA–ssDNA and stimulates RPA ubiquitylation through a process requiring both PRP19 and RFWD3, thereby triggering a phosphorylation-ubiquitylation circuitry that promotes ATR activation and HR.

Cardiac telomere length in heart development, function, and disease.

Telomeres are repetitive nucleoprotein structures at chromosome ends, and a decrease in the number of these repeats, known as a reduction in telomere length (TL), triggers cellular senescence and apoptosis. Heart disease, the worldwide leading cause of death, often results from the loss of cardiac cells, which could be explained by decreases in TL. Due to the cell-specific regulation of TL, this review focuses on studies that have measured telomeres in heart cells and critically assesses the relationship between cardiac TL and heart function. There are several lines of evidence that have identified rapid changes in cardiac TL during the onset and progression of heart disease as well as at critical stages of development. There are also many factors, such as the loss of telomeric proteins, oxidative stress, and hypoxia, that decrease cardiac TL and heart function. In contrast, antioxidants, calorie restriction, and exercise can prevent both cardiac telomere attrition and the progression of heart disease. TL in the heart is also indicative of proliferative potential and could facilitate the identification of cells suitable for cardiac rejuvenation. Although these findings highlight the involvement of TL in heart function, there are important questions regarding the validity of animal models, as well as several confounding factors, that need to be considered when interpreting results and planning future research. With these in mind, elucidating the telomeric mechanisms involved in heart development and the transition to disease holds promise to prevent cardiac dysfunction and potentiate regeneration after injury.

Ubiquitination and SUMOylation in Telomere Maintenance and Dysfunction.

Telomeres are essential nucleoprotein structures at linear chromosomes that maintain genome integrity by protecting chromosome ends from being recognized and processed as damaged DNA. In addition, they limit the cell’s proliferative capacity, as progressive loss of telomeric DNA during successive rounds of cell division eventually causes a state of telomere dysfunction that prevents further cell division. When telomeres become critically short, the cell elicits a DNA damage response resulting in senescence, apoptosis or genomic instability, thereby impacting on aging and tumorigenesis. Over the past years substantial progress has been made in understanding the role of post-translational modifications in telomere-related processes, including telomere maintenance, replication and dysfunction. This review will focus on recent findings that establish an essential role for ubiquitination and SUMOylation at telomeres.

DNA synthesis determines the binding mode of the human mitochondrial single-stranded DNA-binding protein.

Single-stranded DNA-binding proteins (SSBs) play a key role in genome maintenance, binding and organizing single-stranded DNA (ssDNA) intermediates. Multimeric SSBs, such as the human mitochondrial SSB (HmtSSB), present multiple sites to interact with ssDNA, which has been shown in vitro to enable them to bind a variable number of single-stranded nucleotides depending on the salt and protein concentration. It has long been suggested that different binding modes might be used selectively for different functions. To study this possibility, we used optical tweezers to determine and compare the structure and energetics of long, individual HmtSSB–DNA complexes assembled on preformed ssDNA and on ssDNA generated gradually during ‘in situ’ DNA synthesis. We show that HmtSSB binds to preformed ssDNA in two major modes, depending on salt and protein concentration. However, when protein binding was coupled to strand-displacement DNA synthesis, only one of the two binding modes was observed under all experimental conditions. Our results reveal a key role for the gradual generation of ssDNA in modulating the binding mode of a multimeric SSB protein and consequently, in generating the appropriate nucleoprotein structure for DNA synthetic reactions required for genome maintenance.

Changing Direction: From Therapeutic Telomerase Inhibition to Activation?

High telomerase expression is a hallmark of most types of cancer, and, therefore, strategies to inhibit telomerase as an anticancer treatment have been pursuit for many years. In contrast to cancer, short telomeres and lack of telomerase are associated with aging and age-related disease, including heart disease. Consequently, pharmacological telomerase activation can delay aging and also provides cardioprotection. In this viewpoint, we briefly summarized the 2 faces of telomerase in cancer and during aging. We discuss why we see the therapeutic future in telomerase activation rather than its inhibition, for the treatment of cardiac disease and possibly even in the context of cancer. Cardiovascular disease is a worldwide epidemic and represents the most common cause of morbidity and mortality in the western world. Significant advances in health care have led to a steadily rising life expectancy and consequently to a dramatically increased incidence of age-related diseases, such as cancer and heart disease. One of the main reasons for the high incidence of heart failure (HF) is an extremely low regeneration capacity of mammalian cardiomyocytes. Hence, the only effective way to replace lost cardiomyocytes in humans to date, for instance after myocardial infarction, is heart transplantation, which emphasizes the need for novel intervention and prevention strategies Intensive research provided insights into the cellular and molecular mechanisms of aging. Among others (eg, cellular senescence, genomic instability, stem cell exhaustion, mitochondrial dysfunction, and epigenetic alterations), telomere attrition, in particular, has emerged as a main driver of aging and age-related diseases, such as HF.1 Telomeres are dynamic nucleoprotein structures which form a protective cap to conceal the ends of chromosomes from the DNA repair machinery, thus providing chromosomal and cellular integrity.2 However, telomeres are sensitive to aging because telomerase ( TERT [telomere reverse transcriptase]), the enzyme that maintains telomere length, is repressed in …

The telomeric DNA damage response occurs in the absence of chromatin decompaction.

Telomeres are specialized nucleoprotein structures that protect chromosome ends from DNA damage response (DDR) and DNA rearrangements. The telomeric shelterin protein TRF2 suppresses the DDR, and this function has been attributed to its abilities to trigger t-loop formation or prevent massive decompaction and loss of density of telomeric chromatin. Here, we applied stochastic optical reconstruction microscopy (STORM) to measure the sizes and shapes of functional human telomeres of different lengths and dysfunctional telomeres that elicit a DDR. Telomeres have an ovoid appearance with considerable plasticity in shape. Examination of many telomeres demonstrated that depletion of TRF2, TRF1, or both affected the sizes of only a small subset of telomeres. Costaining of telomeres with DDR markers further revealed that the majority of DDR signaling telomeres retained a normal size. Thus, DDR signaling at telomeres does not require decompaction. We propose that telomeres are monitored by the DDR machinery in the absence of telomere expansion and that the DDR is triggered by changes at the molecular level in structure and protein composition.