scholarly journals Alterations of DNA and Chromatin Structures at Telomeres and Genetic Instability in Mouse Cells Defective in DNA Polymerase α

2005 ◽  
Vol 25 (24) ◽  
pp. 11073-11088 ◽  
Author(s):  
Mirai Nakamura ◽  
Akira Nabetani ◽  
Takeshi Mizuno ◽  
Fumio Hanaoka ◽  
Fuyuki Ishikawa
Keyword(s):  
Telomere Length ◽  
Dna Polymerase ◽  
Temperature Sensitive ◽  
Telomeric Dna ◽  
Dna Polymerase Α ◽  
Genome Wide ◽  
Associated Proteins ◽  
Mouse Cells ◽  
The Impact ◽  
Lagging Strand

ABSTRACT Telomere length is controlled by a homeostatic mechanism that involves telomerase, telomere-associated proteins, and conventional replication machinery. Specifically, the coordinated actions of the lagging strand synthesis and telomerase have been argued. Although DNA polymerase α, an enzyme important for the lagging strand synthesis, has been indicated to function in telomere metabolism in yeasts and ciliates, it has not been characterized in higher eukaryotes. Here, we investigated the impact of compromised polymerase α activity on telomeres, using tsFT20 mouse mutant cells harboring a temperature-sensitive polymerase α mutant allele. When polymerase α was temperature-inducibly inactivated, we observed sequential events that included an initial extension of the G-tail followed by a marked increase in the overall telomere length occurring in telomerase-independent and -dependent manners, respectively. These alterations of telomeric DNA were accompanied by alterations of telomeric chromatin structures as revealed by quantitative chromatin immunoprecipitation and immunofluorescence analyses of TRF1 and POT1. Unexpectedly, polymerase α inhibition resulted in a significantly high incidence of Robertsonian chromosome fusions without noticeable increases in other types of chromosomal aberrations. These results indicate that although DNA polymerase α is essential for genome-wide DNA replication, hypomorphic activity leads to a rather specific spectrum of chromosomal abnormality.

scholarly journals The Function of DNA Polymerase α at Telomeric G Tails Is Important for Telomere Homeostasis

2000 ◽  
Vol 20 (3) ◽  
pp. 786-796 ◽  
Author(s):  
Aegina Adams Martin ◽  
Isabelle Dionne ◽  
Raymund J. Wellinger ◽  
Connie Holm
Keyword(s):  
Telomere Length ◽  
Dna Polymerase ◽  
Telomere Maintenance ◽  
Replication Machinery ◽  
Telomeric Dna ◽  
Dna Polymerase Α ◽  
Telomeric Silencing ◽  
Pass Through ◽  
Telomere Lengthening ◽  
Lagging Strand

ABSTRACT Telomere length control is influenced by several factors, including telomerase, the components of telomeric chromatin structure, and the conventional replication machinery. Although known components of the replication machinery can influence telomere length equilibrium, little is known about why mutations in certain replication proteins cause dramatic telomere lengthening. To investigate the cause of telomere elongation in cdc17/pol1 (DNA polymerase α) mutants, we examined telomeric chromatin, as measured by its ability to repress transcription on telomere-proximal genes, and telomeric DNA end structures in pol1-17 mutants. pol1-17 mutants with elongated telomeres show a dramatic loss of the repression of telomere-proximal genes, or telomeric silencing. In addition,cdc17/pol1 mutants grown under telomere-elongating conditions exhibit significant increases in single-stranded character in telomeric DNA but not at internal sequences. The single strandedness is manifested as a terminal extension of the G-rich strand (G tails) that can occur independently of telomerase, suggesting thatcdc17/pol1 mutants exhibit defects in telomeric lagging-strand synthesis. Interestingly, the loss of telomeric silencing and the increase in the sizes of the G tails at the telomeres temporally coincide and occur before any detectable telomere lengthening is observed. Moreover, the G tails observed incdc17/pol1 mutants incubated at the semipermissive temperature appear only when the cells pass through S phase and are processed by the time cells reach G1. These results suggest that lagging-strand synthesis is coordinated with telomerase-mediated telomere maintenance to ensure proper telomere length control.

scholarly journals The Saccharomyces telomere-binding protein Cdc13p interacts with both the catalytic subunit of DNA polymerase α and the telomerase-associated Est1 protein

2000 ◽  
Vol 14 (14) ◽  
pp. 1777-1788 ◽  
Author(s):  
Haiyan Qi ◽  
Virginia A. Zakian
Keyword(s):  
Dna Polymerase ◽  
Binding Protein ◽  
Point Mutations ◽  
Single Strand ◽  
Catalytic Subunit ◽  
Full Length ◽  
Temperature Sensitive ◽  
Dna Polymerase Α ◽  
Telomere Binding Protein

Saccharomyces telomeres consist of ∼350 bp of C1-3A/TG1-3 DNA. Most of this ∼350 bp is replicated by standard, semiconservative DNA replication. After conventional replication, the C1-3A strand is degraded to generate a long single strand TG1-3 tail that can serve as a substrate for telomerase. Cdc13p is a single strand TG1-3DNA-binding protein that localizes to telomeres in vivo. Genetic data suggest that the Cdc13p has multiple roles in telomere replication. We used two hybrid analysis to demonstrate that Cdc13p interacted with both the catalytic subunit of DNA polymerase α, Pol1p, and the telomerase RNA-associated protein, Est1p. The association of these proteins was confirmed by biochemical analysis using full-length or nearly full-length proteins. Point mutations in either CDC13 orPOL1 that reduced the Cdc13p–Pol1p interaction resulted in telomerase mediated telomere lengthening. Over–expression of the carboxyl terminus of Est1p partially suppressed the temperature sensitive lethality of a cdc13-1 strain. We propose that Cdc13p's interaction with Est1p promotes TG1-3 strand lengthening by telomerase and its interaction with Pol1p promotes C1-3A strand resynthesis by DNA polymerase α.

scholarly journals Cell Lysate Microarray for Mapping the Network of Genetic Regulators for Histone Marks

2017 ◽  
Author(s):  
Li Cheng ◽  
Cheng-xi Liu ◽  
Shuang-ying Jiang ◽  
Sha Hou ◽  
Jin-guo Huang ◽  
...  
Keyword(s):  
Temperature Sensitive ◽  
General Applicability ◽  
Post Translational Modifications ◽  
Histone Marks ◽  
Cell Lysate ◽  
Genome Wide ◽  
Protein Functions ◽  
A Cell ◽  
Global Identification ◽  
The Impact

SUMMARYProtein, as the major executer for cell progresses and functions, its abundance and the level of post-translational modifications, are tightly monitored by regulators. Genetic perturbation could help us to understand the relationships between genes and protein functions. Herein, we developed a cell lysate microarray on kilo-conditions (CLICK) from 4,837 yeast knockout (YKO) strains and 322 temperature-sensitive mutant strains to explore the impact of the genome-wide interruption on certain protein. Taking histone marks as examples, a general workflow was established for the global identification of upstream regulators. Through a single CLICK array test, we obtained a series of regulators for H3K4me3 which covers most of the known regulators in Saccharomyces cerevisiae. We also noted that several group of proteins that are linked to negatively regulation of H3K4me3. Further, we discovered that Cab4p and Cab5p, two key enzymes of CoA biosynthesis, play central roles in histone acylation. Because of its general applicability, CLICK array could be easily adopted to rapid and global identification of upstream protein/enzyme(s) that regulate/modify the level of a protein or the posttranslational modification of a non-histone protein.

Positive and negative regulation of telomerase access to the telomere

2000 ◽  
Vol 113 (19) ◽  
pp. 3357-3364 ◽  
Author(s):  
S.K. Evans ◽  
V. Lundblad
Keyword(s):  
Telomere Length ◽  
Mammalian Cells ◽  
Feedback System ◽  
Order Structure ◽  
Telomeric Dna ◽  
Repeat Sequences ◽  
Fundamental Part ◽  
Telomere Function ◽  
Associated Proteins ◽  
Chromosome Integrity

The protective caps on chromosome ends - known as telomeres - consist of DNA and associated proteins that are essential for chromosome integrity. A fundamental part of ensuring proper telomere function is maintaining adequate length of the telomeric DNA tract. Telomeric repeat sequences are synthesized by the telomerase reverse transcriptase, and, as such, telomerase is a central player in the maintenance of steady-state telomere length. Evidence from both yeast and mammals suggests that telomere-associated proteins positively or negatively control access of telomerase to the chromosome terminus. In yeast, positive regulation of telomerase access appears to be achieved through recruitment of the enzyme by the end-binding protein Cdc13p. In contrast, duplex-DNA-binding proteins assembled along the telomeric tract exert a feedback system that negatively modulates telomere length by limiting the action of telomerase. In mammalian cells, and perhaps also in yeast, binding of these proteins probably promotes a higher-order structure that renders the telomere inaccessible to the telomerase enzyme.

scholarly journals Separable, Ctf4-mediated recruitment of DNA Polymerase α for initiation of DNA synthesis at replication origins and lagging-strand priming during replication elongation

PLoS Genetics ◽  
2020 ◽  
Vol 16 (5) ◽  
pp. e1008755 ◽  
Author(s):  
Sarina Y. Porcella ◽  
Natasha C. Koussa ◽  
Colin P. Tang ◽  
Daphne N. Kramer ◽  
Priyanka Srivastava ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Replication Origins ◽  
Dna Polymerase Α ◽  
Lagging Strand

scholarly journals Processing of eukaryotic Okazaki fragments by redundant nucleases can be uncoupled from ongoing DNA replicationin vivo

2018 ◽  
Author(s):  
Malik Kahli ◽  
Joseph S. Osmundson ◽  
Rani Yeung ◽  
Duncan J. Smith
Keyword(s):  
Dna Polymerase ◽  
Systematic Evaluation ◽  
Additional Contribution ◽  
Strand Displacement ◽  
Dna Polymerase Delta ◽  
Okazaki Fragment ◽  
Okazaki Fragments ◽  
The Impact ◽  
Lagging Strand

ABSTRACTPrior to ligation, each Okazaki fragment synthesized on the lagging strand in eukaryotes must be nucleolytically processed. Nuclease cleavage takes place in the context of 5’ flap structures generated via strand-displacement synthesis by DNA polymerase delta. At least three DNA nucleases: Rad27 (Fen1), Dna2, and Exo1, have been implicated in processing Okazaki fragment flaps. However, neither the contributions of individual nucleases to lagging-strand synthesis nor the structure of the DNA intermediates formed in their absence have been clearly definedin vivo.By conditionally depleting lagging-strand nucleases and directly analyzing Okazaki fragments synthesizedin vivoinS. cerevisiae, we conduct a systematic evaluation of the impact of Rad27, Dna2 and Exo1 on lagging-strand synthesis. We find that Rad27 processes the majority of lagging-strand flaps, with a significant additional contribution from Exo1 but not from Dna2. When nuclease cleavage is impaired, we observe a reduction in strand-displacement synthesis as opposed to the widespread generation of long Okazaki fragment 5’ flaps, as predicted by some models. Further, using cell cycle-restricted constructs, we demonstrate that both the nucleolytic processing and the ligation of Okazaki fragments can be uncoupled from DNA replication and delayed until after synthesis of the majority of the genome is complete.

scholarly journals Mechanism for priming DNA synthesis by yeast DNA Polymerase α

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Rajika L Perera ◽  
Rubben Torella ◽  
Sebastian Klinge ◽  
Mairi L Kilkenny ◽  
Joseph D Maman ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Catalytic Core ◽  
Dna Polymerase Α ◽  
Dna Oligonucleotides ◽  
Computational Data ◽  
Dna Template ◽  
Lagging Strand ◽  
Primase Complex ◽  
Nucleotide Polymerization

The DNA Polymerase α (Pol α)/primase complex initiates DNA synthesis in eukaryotic replication. In the complex, Pol α and primase cooperate in the production of RNA-DNA oligonucleotides that prime synthesis of new DNA. Here we report crystal structures of the catalytic core of yeast Pol α in unliganded form, bound to an RNA primer/DNA template and extending an RNA primer with deoxynucleotides. We combine the structural analysis with biochemical and computational data to demonstrate that Pol α specifically recognizes the A-form RNA/DNA helix and that the ensuing synthesis of B-form DNA terminates primer synthesis. The spontaneous release of the completed RNA-DNA primer by the Pol α/primase complex simplifies current models of primer transfer to leading- and lagging strand polymerases. The proposed mechanism of nucleotide polymerization by Pol α might contribute to genomic stability by limiting the amount of inaccurate DNA to be corrected at the start of each Okazaki fragment.

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