scholarly journals Structure of human CST-pol-α/primase bound to a telomeric overhang poised for initiation of telomere C-strand synthesis

2021 ◽  
Author(s):  
Qixiang He ◽  
Xiuhua Lin ◽  
Bianca L Chavez ◽  
Benjamin L Lusk ◽  
Ci Ji Lim
Keyword(s):  
Genome Stability ◽  
De Novo ◽  
Protein A ◽  
Telomere Maintenance ◽  
Structural Basis ◽  
Particle Analysis ◽  
Single Stranded Dna ◽  
Dna And Rna ◽  
Exonuclease Domain ◽  
Dna Template

Telomere replication and regulation protect mammalian chromosome ends and promote genome stability. An essential step in telomere maintenance is the C-strand fill-in process, which is the de novo synthesis of the complementary strand of the telomere overhang. This step is catalyzed by polymerase-alpha/primase complex (pol-α/primase) and coordinated by an accessory factor, CTC1-STN1-TEN1 (CST). Using cryogenic-electron microscopy single-particle analysis, we report the structure of the human telomere C-strand fill-in preinitiation complex (PIC) at 3.9 Å resolution. The structure reveals a CST and a pol-α/primase co-bound to a single telomere overhang, poised for de novo RNA primer synthesis. Upon PIC assembly, the pol-α/primase undergoes large conformation change from its apo-state; CST partitions the DNA and RNA catalytic centers of pol-α/primase into two separate domains and positions the 3' end of an extended telomere single-stranded DNA template towards the RNA catalytic center (PRIM1 or p49). The telomeric single-stranded DNA template is further positioned by the POLA1 (or p180) catalytically dead exonuclease domain. Together with CST, the exonuclease domain forms a tight-fit molecular tunnel for template direction. Given the structural homology of CST to Replication Protein A (RPA), our structure provides the structural basis for a new model of how pol-α/primase lagging-strand DNA synthesis is coordinated by single-stranded DNA-binding accessory factors.

scholarly journals The cryo-EM structure of human CST reveals a two-megadalton decameric assembly bound to telomeric DNA

2019 ◽  
Author(s):  
Ci Ji Lim ◽  
Alexandra T. Barbour ◽  
Arthur J. Zaug ◽  
Allison E. McKay ◽  
Deborah S. Wuttke ◽  
...  
Keyword(s):  
Dna Binding ◽  
Genome Stability ◽  
De Novo ◽  
Protein A ◽  
Telomere Maintenance ◽  
Telomeric Dna ◽  
Single Stranded Dna ◽  
Recovery Processes ◽  
Resolution Model ◽  
Architectural Organization

AbstractThe single-stranded DNA-binding CTC1-STN1-TEN1 (CST) complex is essential for telomere maintenance and genome-wide replication recovery, processes that are critical for genome stability. Here, we report the 2.95 Å cryo-EM structure of human CST bound to telomeric single-stranded DNA, which unexpectedly assembles as a decameric supercomplex. The atomic model of the 134 kDa CTC1, built almost entirely de novo, reveals the overall architecture of CST and the DNA-binding anchor site. In situ arrangements of STN1 and TEN1 are revealed, with STN1 interacting with CTC1 at two separated sites, allowing allosteric mediation of CST decameric assembly. Surprisingly, CTC1 lacks the anticipated structural homology to yeast Cdc13 but instead shares similarity with a form of Replication Protein A. The atomic-resolution model of human CST provides crucial mechanistic understanding of CST mutations associated with human diseases. Moreover, the decameric form of CST suggests the intriguing possibility of ssDNA architectural organization similar to what the nucleosome provides for dsDNA.One Sentence SummaryHuman telomeric single-stranded DNA triggers the assembly of a decameric protein supercomplex solved by cryo-EM.

scholarly journals Trf1 Is Not Required for Proliferation or Functional Telomere Maintenance in Chicken DT40 Cells

2009 ◽  
Vol 20 (10) ◽  
pp. 2563-2571 ◽  
Author(s):  
Carol Cooley ◽  
Katie M. Baird ◽  
Virginie Faure ◽  
Thomas Wenner ◽  
Jillian L. Stewart ◽  
...  
Keyword(s):  
Genome Stability ◽  
De Novo ◽  
Telomere Maintenance ◽  
Clonal Variation ◽  
Fish Analysis ◽  
Double Stranded Dna ◽  
Late Passage ◽  
Telomere Structure ◽  
Dt40 Cells ◽  
Novel Exon

The telomere end-protection complex prevents the ends of linear eukaryotic chromosomes from degradation or inappropriate DNA repair. The homodimeric double-stranded DNA-binding protein, Trf1, is a component of this complex and is essential for mouse embryonic development. To define the requirement for Trf1 in somatic cells, we deleted Trf1 in chicken DT40 cells by gene targeting. Trf1-deficient cells proliferated as rapidly as control cells and showed telomeric localization of Trf2, Rap1, and Pot1. Telomeric G-strand overhang lengths were increased in late-passage Trf1-deficient cells, although telomere lengths were unaffected by Trf1 deficiency, as determined by denaturing Southern and quantitative FISH analysis. Although we observed some clonal variation in terminal telomere fragment lengths, this did not correlate with cellular Trf1 levels. Trf1 was not required for telomere seeding, indicating that de novo telomere formation can proceed without Trf1. The Pin2 isoform and a novel exon 4, 5–deleted isoform localized to telomeres in Trf1-deficient cells. Trf1-deficient cells were sensitive to DNA damage induced by ionizing radiation. Our data demonstrate that chicken DT40 B cells do not require Trf1 for functional telomere structure and suggest that Trf1 may have additional, nontelomeric roles involved in maintaining genome stability.

scholarly journals The structure of human CST reveals a decameric assembly bound to telomeric DNA

Science ◽  
2020 ◽  
Vol 368 (6495) ◽  
pp. 1081-1085 ◽  
Author(s):  
Ci Ji Lim ◽  
Alexandra T. Barbour ◽  
Arthur J. Zaug ◽  
Karen J. Goodrich ◽  
Allison E. McKay ◽  
...  
Keyword(s):  
De Novo ◽  
Protein A ◽  
Structural Similarity ◽  
Telomere Maintenance ◽  
Telomeric Dna ◽  
Double Stranded Dna ◽  
Cryo Electron Microscopy ◽  
Genome Wide ◽  
Carboxyl Terminal ◽  
Stalled Replication Forks

The CTC1-STN1-TEN1 (CST) complex is essential for telomere maintenance and resolution of stalled replication forks genome-wide. Here, we report the 3.0-angstrom cryo–electron microscopy structure of human CST bound to telomeric single-stranded DNA (ssDNA), which assembles as a decameric supercomplex. The atomic model of the 134-kilodalton CTC1 subunit, built almost entirely de novo, reveals the overall architecture of CST and the DNA-binding anchor site. The carboxyl-terminal domain of STN1 interacts with CTC1 at two separate docking sites, allowing allosteric mediation of CST decamer assembly. Furthermore, ssDNA appears to staple two monomers to nucleate decamer assembly. CTC1 has stronger structural similarity to Replication Protein A than the expected similarity to yeast Cdc13. The decameric structure suggests that CST can organize ssDNA analogously to the nucleosome’s organization of double-stranded DNA.

Defective Telomerase in Familial Myelodysplasia and Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 849-849
Author(s):  
Michael Kirwan ◽  
Tom Vulliamy ◽  
Amanda J. Walne ◽  
Richard Beswick ◽  
Peter Hillmen ◽  
...  
Keyword(s):  
Myeloid Leukemia ◽  
De Novo ◽  
Protein A ◽  
Telomerase Activity ◽  
Telomere Maintenance ◽  
Diverse Range ◽  
Enhancer Binding Protein ◽  
Binding Factor ◽  
Risk Of Progression

Abstract Myelodysplastic syndromes (MDS) comprise a diverse range of hematological conditions, all of which involve impaired or disturbed hematopoiesis, cytopenias, marrow dysplasia and the risk of progression to acute myeloid leukemia (AML). MDS and AML are believed to involve a multi-step process arising within bone marrow stem cells, but the specific defects responsible remain poorly understood. In cases of inherited, familial forms of the disease two genes have been implicated: the hematopoietic transcription factors CBFA2 (core binding factor a2) and CEBPA (CCATT box enhancer binding protein a) and these account for only a small number of the familial cases so far reported. The uncharacterized families represent an ideal resource for identifying the primary pathology of the disease. In this study we have screened 21 families where MDS/AML appears to be familial for mutations in the telomerase enzyme components TERC (telomerase RNA component) and TERT (telomerase reverse transcriptase). In two families, we have identified novel mutations in TERC and in another two we have identified mutations in TERT directly resulting in MDS/AML. Functional analysis has demonstrated that all mutations adversely impact telomerase activity in vitro and affected individuals have short telomeres (see Table 1 for summary). These results have several important implications: First, they identify a novel genetic pathway (defective telomere maintenance) that can lead to familial MDS/AML; second, they suggest that TERC and TERT act as tumour suppressors and; finally, they have implications for the biology, treatment and screening regimen for de novo cases of MDS/AML. Family Number of affected individuals Mutation Telomerase activity compared to wild type Age-adjusted telomere length 1 3 TERC c.212C>G 1% −4.62 2 5 TERC c.309G>T 4% 0.01 3 2 TERTc.1892G>A 0% −4.45 4 2 TERTc.2354C>T 11% −3.12 Table 1 – Summary of clinical and molecular analyses of four families with MDS/AML

scholarly journals Human replication protein A induces dynamic changes in single-stranded DNA and RNA structures

2019 ◽  
Vol 294 (38) ◽  
pp. 13915-13927 ◽  
Author(s):  
Qing-Man Wang ◽  
Yan-Tao Yang ◽  
Yi-Ran Wang ◽  
Bo Gao ◽  
Xuguang Xi ◽  
...  
Keyword(s):  
Protein A ◽  
Replication Protein A ◽  
Replication Protein ◽  
Rna Structures ◽  
Dynamic Changes ◽  
Single Stranded Dna ◽  
Dna And Rna

PrimPol (Primase/Polymerase), replicating enzyme – A mini review

2020 ◽  
Vol 2 (4) ◽  
pp. 89-92
Author(s):  
Muhammad Amir ◽  
Sabeera Afzal ◽  
Alia Ishaq
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Genetic Information ◽  
Nuclear Dna ◽  
De Novo ◽  
Dna Polymerases ◽  
Test Site ◽  
Mitochondrial Dna Replication ◽  
Dna Template ◽  
Preliminary Phase

Polymerases were revealed first in 1970s. Most important to the modest perception the enzyme responsible for nuclear DNA replication that was pol , for DNA repair pol and for mitochondrial DNA replication pol  DNA construction and renovation done by DNA polymerases, so directing both the constancy and discrepancy of genetic information. Replication of genome initiate with DNA template-dependent fusion of small primers of RNA. This preliminary phase in replication of DNA demarcated as de novo primer synthesis which is catalyzed by specified polymerases known as primases. Sixteen diverse DNA-synthesizing enzymes about human perspective are devoted to replication, reparation, mutilation lenience, and inconsistency of nuclear DNA. But in dissimilarity, merely one DNA polymerase has been called in mitochondria. It has been suggest that PrimPol is extremely acting the roles by re-priming DNA replication in mitochondria to permit an effective and appropriate way replication to be accomplished. Investigations from a numeral of test site have significantly amplified our appreciative of the role, recruitment and regulation of the enzyme during DNA replication. Though, we are simply just start to increase in value the versatile roles that play PrimPol in eukaryote.

scholarly journals RAD50 and RAD51 Define Two Pathways That Collaborate to Maintain Telomeres in the Absence of Telomerase

Genetics ◽  
1999 ◽  
Vol 152 (1) ◽  
pp. 143-152 ◽  
Author(s):  
Siyuan Le ◽  
J Kent Moore ◽  
James E Haber ◽  
Carol W Greider
Keyword(s):  
Cell Death ◽  
Telomere Length ◽  
Gene Conversion ◽  
De Novo ◽  
Dna Recombination ◽  
Telomere Maintenance ◽  
Triple Mutant ◽  
Yeast Telomerase ◽  
Telomere Lengthening

Abstract Telomere length is maintained by the de novo addition of telomere repeats by telomerase, yet recombination can elongate telomeres in the absence of telomerase. When the yeast telomerase RNA component, TLC1, is deleted, telomeres shorten and most cells die. However, gene conversion mediated by the RAD52 pathway allows telomere lengthening in rare survivor cells. To further investigate the role of recombination in telomere maintenance, we assayed telomere length and the ability to generate survivors in several isogenic DNA recombination mutants, including rad50, rad51, rad52, rad54, rad57, xrs2, and mre11. The rad51, rad52, rad54, and rad57 mutations increased the rate of cell death in the absence of TLC1. In contrast, although the rad50, xrs2, and mre11 strains initially had short telomeres, double mutants with tlc1 did not affect the rate of cell death, and survivors were generated at later times than tlc1 alone. While none of the double mutants of recombination genes and tlc1 (except rad52 tlc1) blocked the ability to generate survivors, a rad50 rad51 tlc1 triple mutant did not allow the generation of survivors. Thus RAD50 and RAD51 define two separate pathways that collaborate to allow cells to survive in the absence of telomerase.

scholarly journals The Yeast Recombinational Repair Protein Rad59 Interacts With Rad52 and Stimulates Single-Strand Annealing

Genetics ◽  
2001 ◽  
Vol 159 (2) ◽  
pp. 515-525 ◽  
Author(s):  
Allison P Davis ◽  
Lorraine S Symington
Keyword(s):  
Protein A ◽  
Single Strand ◽  
Physical Interaction ◽  
Dna Double Strand Breaks ◽  
Single Stranded Dna ◽  
Single Strand Annealing ◽  
Strand Annealing ◽  
Recombination Pathway

Abstract The yeast RAD52 gene is essential for homology-dependent repair of DNA double-strand breaks. In vitro, Rad52 binds to single- and double-stranded DNA and promotes annealing of complementary single-stranded DNA. Genetic studies indicate that the Rad52 and Rad59 proteins act in the same recombination pathway either as a complex or through overlapping functions. Here we demonstrate physical interaction between Rad52 and Rad59 using the yeast two-hybrid system and co-immunoprecipitation from yeast extracts. Purified Rad59 efficiently anneals complementary oligonucleotides and is able to overcome the inhibition to annealing imposed by replication protein A (RPA). Although Rad59 has strand-annealing activity by itself in vitro, this activity is insufficient to promote strand annealing in vivo in the absence of Rad52. The rfa1-D288Y allele partially suppresses the in vivo strand-annealing defect of rad52 mutants, but this is independent of RAD59. These results suggest that in vivo Rad59 is unable to compete with RPA for single-stranded DNA and therefore is unable to promote single-strand annealing. Instead, Rad59 appears to augment the activity of Rad52 in strand annealing.

scholarly journals Characterization of metabolic responses, genetic variations, and microsatellite instability in ammonia-stressed CHO cells grown in fed-batch cultures

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Dylan G. Chitwood ◽  
Qinghua Wang ◽  
Kathryn Elliott ◽  
Aiyana Bullock ◽  
Dwon Jordana ◽  
...  
Keyword(s):  
Microsatellite Instability ◽  
Microsatellite Loci ◽  
Cho Cells ◽  
Genome Stability ◽  
De Novo ◽  
Genome Instability ◽  
Chinese Hamster ◽  
Functional Genes ◽  
Nucleotide Polymorphisms ◽  
De Novo Mutations

Abstract Background As bioprocess intensification has increased over the last 30 years, yields from mammalian cell processes have increased from 10’s of milligrams to over 10’s of grams per liter. Most of these gains in productivity can be attributed to increasing cell densities within bioreactors. As such, strategies have been developed to minimize accumulation of metabolic wastes, such as lactate and ammonia. Unfortunately, neither cell growth nor biopharmaceutical production can occur without some waste metabolite accumulation. Inevitably, metabolic waste accumulation leads to decline and termination of the culture. While it is understood that the accumulation of these unwanted compounds imparts a suboptimal culture environment, little is known about the genotoxic properties of these compounds that may lead to global genome instability. In this study, we examined the effects of high and moderate extracellular ammonia on the physiology and genomic integrity of Chinese hamster ovary (CHO) cells. Results Through whole genome sequencing, we discovered 2394 variant sites within functional genes comprised of both single nucleotide polymorphisms and insertion/deletion mutations as a result of ammonia stress with high or moderate impact on functional genes. Furthermore, several of these de novo mutations were found in genes whose functions are to maintain genome stability, such as Tp53, Tnfsf11, Brca1, as well as Nfkb1. Furthermore, we characterized microsatellite content of the cultures using the CriGri-PICR Chinese hamster genome assembly and discovered an abundance of microsatellite loci that are not replicated faithfully in the ammonia-stressed cultures. Unfaithful replication of these loci is a signature of microsatellite instability. With rigorous filtering, we found 124 candidate microsatellite loci that may be suitable for further investigation to determine whether these loci may be reliable biomarkers to predict genome instability in CHO cultures. Conclusion This study advances our knowledge with regards to the effects of ammonia accumulation on CHO cell culture performance by identifying ammonia-sensitive genes linked to genome stability and lays the foundation for the development of a new diagnostic tool for assessing genome stability.

scholarly journals Cells Expressing Murine RAD52 Splice Variants Favor Sister Chromatid Repair

2006 ◽  
Vol 26 (10) ◽  
pp. 3752-3763 ◽  
Author(s):  
Peter H. Thorpe ◽  
Vanessa A. Marrero ◽  
Margaret H. Savitzky ◽  
Ivana Sunjevaric ◽  
Tom C. Freeman ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Sister Chromatid ◽  
Mammalian Cells ◽  
Splice Variants ◽  
Protein A ◽  
Single Stranded Dna ◽  
Culture Cells ◽  
Dominant Phenotype ◽  
Mouse Tissues

ABSTRACT The RAD52 gene is essential for homologous recombination in the yeast Saccharomyces cerevisiae. RAD52 is the archetype in an epistasis group of genes essential for DNA damage repair. By catalyzing the replacement of replication protein A with Rad51 on single-stranded DNA, Rad52 likely promotes strand invasion of a double-stranded DNA molecule by single-stranded DNA. Although the sequence and in vitro functions of mammalian RAD52 are conserved with those of yeast, one difference is the presence of introns and consequent splicing of the mammalian RAD52 pre-mRNA. We identified two novel splice variants from the RAD52 gene that are expressed in adult mouse tissues. Expression of these splice variants in tissue culture cells elevates the frequency of recombination that uses a sister chromatid template. To characterize this dominant phenotype further, the RAD52 gene from the yeast Saccharomyces cerevisiae was truncated to model the mammalian splice variants. The same dominant sister chromatid recombination phenotype seen in mammalian cells was also observed in yeast. Furthermore, repair from a homologous chromatid is reduced in yeast, implying that the choice of alternative repair pathways may be controlled by these variants. In addition, a dominant DNA repair defect induced by one of the variants in yeast is suppressed by overexpression of RAD51, suggesting that the Rad51-Rad52 interaction is impaired.

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