scholarly journals Detection of Base Analogs Incorporated During DNA Replication by Nanopore Sequencing

2019 ◽  
Author(s):  
Daniela Georgieva ◽  
Qian Liu ◽  
Kai Wang ◽  
Dieter Egli
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Mammalian Cells ◽  
Cell Transformation ◽  
Ionic Current ◽  
Stem Cell Differentiation ◽  
Synthetic Dna ◽  
Dna Strands ◽  
Sequencing Platforms ◽  
Thymidine Analogs

ABSTRACTDNA synthesis is a fundamental requirement for cell proliferation and DNA repair, but no tools exist to identify the location, direction and speed of replication forks with base pair resolution. Mammalian cells have the ability to incorporate thymidine analogs along with the natural A, T, G and C bases during DNA synthesis, which allows for labelling of replicating or repaired DNA. Most sequencing platforms rely on base-pairing to identify the four canonical nucleotides, and are thus unable to distinguish them from these analogs. In contrast, the Oxford Nanopore Technologies (ONT) MinION infers nucleotide identity from changes in the ionic current as DNA strands are pulled through nanopores and can in principle differentiate noncanonical nucleotides from natural ones. Here, we demonstrate the use of the ONT MinION to detect 11 different thymidine analogs including CldU, BrdU, IdU, as well as, EdU alone or coupled to Biotin and other bulky adducts in synthetic DNA templates. We also show detection of IdU in the genome of mouse pluripotent stem cells. We find that different modifications generate variable shifts in ionic signals, providing a method of using analog combinations to identify the location and direction of DNA synthesis and repair at high resolution. We conclude that this novel method has the potential for single-base, genome-wide examination of DNA replication in stem cell differentiation or cell transformation.

scholarly journals Detection of base analogs incorporated during DNA replication by nanopore sequencing

2020 ◽  
Vol 48 (15) ◽  
pp. e88-e88 ◽  
Author(s):  
Daniela Georgieva ◽  
Qian Liu ◽  
Kai Wang ◽  
Dieter Egli
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Mammalian Cells ◽  
Single Dna Molecules ◽  
Synthetic Dna ◽  
Oxford Nanopore ◽  
Wide Range ◽  
Dna Strands ◽  
Single Method ◽  
Thymidine Analogs

Abstract DNA synthesis is a fundamental requirement for cell proliferation and DNA repair, but no single method can identify the location, direction and speed of replication forks with high resolution. Mammalian cells have the ability to incorporate thymidine analogs along with the natural A, T, G and C bases during DNA synthesis, which allows for labeling of replicating or repaired DNA. Here, we demonstrate the use of the Oxford Nanopore Technologies MinION to detect 11 different thymidine analogs including CldU, BrdU, IdU as well as EdU alone or coupled to Biotin and other bulky adducts in synthetic DNA templates. We also show that the large adduct Biotin can be distinguished from the smaller analog IdU, which opens the possibility of using analog combinations to identify the location and direction of DNA synthesis. Furthermore, we detect IdU label on single DNA molecules in the genome of mouse pluripotent stem cells and using CRISPR/Cas9-mediated enrichment, determine replication rates using newly synthesized DNA strands in human mitochondrial DNA. We conclude that this novel method, termed Replipore sequencing, has the potential for on target examination of DNA replication in a wide range of biological contexts.

DNA replication and repair of Tilapia cells. II. Effects of temperature on DNA replication and ultraviolet repair in Tilapia ovary cells

1988 ◽  
Vol 89 (2) ◽  
pp. 263-272
Author(s):  
J.D. Chen ◽  
F.H. Yew
Keyword(s):  
Molecular Weight ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Mammalian Cells ◽  
Excision Repair ◽  
Control Level ◽  
High Molecular Weight Dna ◽  
Wide Range ◽  
Effects Of Temperature ◽  
Fish Cells

TO-2 is a fish cell line derived from the Tilapia ovary. It grows over a wide range of temperature (15–34 degrees C). While most fish cells lack DNA excision repair and are hypersensitive to ultraviolet light (u.v.), Tilapia cells are more u.v.-resistant than mammalian cells. In this paper we report the effects of temperature on DNA replication and u.v. repair in TO-2 cells. When the cells were moved from 31 degrees C to the sublethal high temperature of 37 degrees C, the rate of DNA synthesis first decreased to 60%, then speedy recovery soon set in, and after 8 h at 37 degrees C the rate of DNA synthesis overshot the 31 degrees C control level by 180%. When moved to low temperature (18 degrees C) Tilapia cells also showed an initial suppression of DNA synthesis before settling at 30% of the control level. u.v. reduced but could not block DNA synthesis completely. The inhibition was overcome in 3 h at 37, 31 and 25 degrees C, but not at 18 degrees C. Initiation of nascent DNA synthesis was blocked at 4 J m-2 in TO-2 cells compared with less than or equal to 1 J m-2 in mammalian cells. After 9 J m-2 u.v. irradiation, low molecular weight DNA replication intermediates started to accumulate, and they could be chased into high molecular weight DNA with little delay. TO-2 cells showed low levels of u.v.-induced excision repair; but this was prominent compared with other fish cells. The u.v.-induced incision rate has been measured at various temperatures, and the activation energy of incision estimated to be 13 kcal mol-1 (1 cal approximately equal to 4.184 J).

scholarly journals Nonperiodic Activity of the Human Anaphase-Promoting Complex–Cdh1 Ubiquitin Ligase Results in Continuous DNA Synthesis Uncoupled from Mitosis

2000 ◽  
Vol 20 (20) ◽  
pp. 7613-7623 ◽  
Author(s):  
Claus Storgaard Sørensen ◽  
Claudia Lukas ◽  
Edgar R. Kramer ◽  
Jan-Michael Peters ◽  
Jiri Bartek ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Ubiquitin Ligase ◽  
Mammalian Cells ◽  
Kinase Inhibitor ◽  
Cyclin B1 ◽  
S Phase ◽  
Anaphase Promoting Complex

ABSTRACT Ubiquitin-proteasome-mediated destruction of rate-limiting proteins is required for timely progression through the main cell cycle transitions. The anaphase-promoting complex (APC), periodically activated by the Cdh1 subunit, represents one of the major cellular ubiquitin ligases which, in Saccharomyces cerevisiae andDrosophila spp., triggers exit from mitosis and during G1 prevents unscheduled DNA replication. In this study we investigated the importance of periodic oscillation of the APC-Cdh1 activity for the cell cycle progression in human cells. We show that conditional interference with the APC-Cdh1 dissociation at the G1/S transition resulted in an inability to accumulate a surprisingly broad range of critical mitotic regulators including cyclin B1, cyclin A, Plk1, Pds1, mitosin (CENP-F), Aim1, and Cdc20. Unexpectedly, although constitutively assembled APC-Cdh1 also delayed G1/S transition and lowered the rate of DNA synthesis during S phase, some of the activities essential for DNA replication became markedly amplified, mainly due to a progressive increase of E2F-dependent cyclin E transcription and a rapid turnover of the p27Kip1 cyclin-dependent kinase inhibitor. Consequently, failure to inactivate APC-Cdh1 beyond the G1/S transition not only inhibited productive cell division but also supported slow but uninterrupted DNA replication, precluding S-phase exit and causing massive overreplication of the genome. Our data suggest that timely oscillation of the APC-Cdh1 ubiquitin ligase activity represents an essential step in coordinating DNA replication with cell division and that failure of mechanisms regulating association of APC with the Cdh1 activating subunit can undermine genomic stability in mammalian cells.

scholarly journals Two New Early Bacteriophage T4 Genes,repEA and repEB, That Are Important for DNA Replication Initiated from Origin E

1999 ◽  
Vol 181 (22) ◽  
pp. 7115-7125 ◽  
Author(s):  
Rita Vaiskunaite ◽  
Andrew Miller ◽  
Laura Davenport ◽  
Gisela Mosig
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Bacteriophage T4 ◽  
Direct Synthesis ◽  
Growth Conditions ◽  
Dna Strands ◽  
Different Temperatures ◽  
And Function ◽  
Different Origins

ABSTRACT Two new, small, early bacteriophage T4 genes, repEA andrepEB, located within the origin E (oriE) region of T4 DNA replication, affect functioning of this origin. An important and unusual property of the oriE region is that it is transcribed at early and late periods after infection, but in opposite directions (from complementary DNA strands). The early transcripts are mRNAs for RepEA and RepEB proteins, and they can serve as primers for leading-strand DNA synthesis. The late transcripts, which are genuine antisense RNAs for the early transcripts, direct synthesis of virion components. Because the T4 genome contains several origins, and because recombination can bypass a primase requirement for retrograde synthesis, neither defects in a single origin nor primase deficiencies are lethal in T4 (Mosig et al., FEMS Microbiol. Rev. 17:83–98, 1995). Therefore, repEA and repEBwere expected and found to be important for T4 DNA replication only when activities of other origins were reduced. To investigate the in vivo roles of the two repE genes, we constructed nonsense mutations in each of them and combined them with the motAmutation sip1 that greatly reduces initiation from other origins. As expected, T4 DNA synthesis and progeny production were severely reduced in the double mutants as compared with the singlemotA mutant, but early transcription of oriEwas reduced neither in the motA nor in the repEmutants. Moreover, residual DNA replication and growth of the double mutants were different at different temperatures, suggesting different functions for repEA and repEB. We surmise that the different structures and protein requirements for functioning of the different origins enhance the flexibility of T4 to adapt to varied growth conditions, and we expect that different origins in other organisms with multiorigin chromosomes might differ in structure and function for similar reasons.

scholarly journals When DNA Polymerases Multitask: Functions Beyond Nucleotidyl Transfer

2022 ◽  
Vol 8 ◽  
Author(s):  
Denisse Carvajal-Maldonado ◽  
Lea Drogalis Beckham ◽  
Richard D. Wood ◽  
Sylvie Doublié
Keyword(s):  
Dna Repair ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Extracurricular Activities ◽  
Damage Tolerance ◽  
Dna Polymerases ◽  
Translesion Dna Synthesis ◽  
Dna Strands ◽  
Translesion Dna ◽  
Central Reaction

DNA polymerases catalyze nucleotidyl transfer, the central reaction in synthesis of DNA polynucleotide chains. They function not only in DNA replication, but also in diverse aspects of DNA repair and recombination. Some DNA polymerases can perform translesion DNA synthesis, facilitating damage tolerance and leading to mutagenesis. In addition to these functions, many DNA polymerases conduct biochemically distinct reactions. This review presents examples of DNA polymerases that carry out nuclease (3ʹ—5′ exonuclease, 5′ nuclease, or end-trimming nuclease) or lyase (5′ dRP lyase) extracurricular activities. The discussion underscores how DNA polymerases have a remarkable ability to manipulate DNA strands, sometimes involving relatively large intramolecular movement.

A human nuclear protein with sequence homology to a family of early S phase proteins is required for entry into S phase and for cell division

1994 ◽  
Vol 107 (1) ◽  
pp. 253-265 ◽  
Author(s):  
I.T. Todorov ◽  
R. Pepperkok ◽  
R.N. Philipova ◽  
S.E. Kearsey ◽  
W. Ansorge ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Amino Acid ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Mammalian Cells ◽  
Nuclear Protein ◽  
Amino Acid Level ◽  
S Phase ◽  
Significant Similarity

Molecular cloning and characterisation of a human nuclear protein designated BM28 is reported. On the amino acid level this 892 amino acid protein, migrating on SDS-gels as a 125 kDa polypeptide, shares areas of significant similarity with a recently defined family of early S phase proteins. The members of this family, the Saccharomyces cerevisiae Mcm2p, Mcm3p, Cdc46p/Mcm5p, the Schizosaccharomyces pombe Cdc21p and the mouse protein P1 are considered to be involved in the onset of DNA replication. The highest similarity was found with Mcm2p (42% identity over the whole length and higher than 75% over a conservative region of 215 amino acid residues), suggesting that BM28 could represent the human homologue of the S. cerevisiae MCM2. Using antibodies raised against the recombinant BM28 the corresponding antigen was found to be localised in the nuclei of various mammalian cells. Microinjection of anti-BM28 antibody into synchronised mouse NIH3T3 or human HeLa cells presents evidence for the involvement of the protein in cell cycle progression. When injected in G1 phase the anti-BM28 antibody inhibits the onset of subsequent DNA synthesis as tested by the incorporation of bromodeoxyuridine. Microinjection during the S phase had no effect on DNA synthesis, but inhibits cell division. The data suggest that the nuclear protein BM28 is required for two events of the cell cycle, for the onset of DNA replication and for cell division.

scholarly journals Genetic Confirmation that the H5 Protein Is Required for Vaccinia Virus DNA Replication

2015 ◽  
Vol 89 (12) ◽  
pp. 6312-6327 ◽  
Author(s):  
Kathleen A. Boyle ◽  
Matthew D. Greseth ◽  
Paula Traktman
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Cell Line ◽  
Mammalian Cells ◽  
Indirect Evidence ◽  
Chemical Mutagenesis ◽  
Temperature Sensitive ◽  
Genome Replication ◽  
Content Type ◽  
Physical Autonomy

ABSTRACTThe duplication of the poxvirus double-stranded DNA genome occurs in cytoplasmic membrane-delimited factories. This physical autonomy from the host nucleus suggests that poxvirus genomes encode the full repertoire of proteins committed for genome replication. Biochemical and genetic analyses have confirmed that six viral proteins are required for efficient DNA synthesis; indirect evidence has suggested that the multifunctional H5 protein may also have a role. Here we show that H5 localizes to replication factories, as visualized by immunofluorescence and immunoelectron microscopy, and can be retrieved upon purification of the viral polymerase holoenzyme complex. The temperature-sensitive (ts) mutant Dts57, which was generated by chemical mutagenesis and has a lesion in H5, exhibits defects in DNA replication and morphogenesis under nonpermissive conditions, depending upon the experimental protocol. The H5 variant encoded by the genome of this mutant istsfor function but not stability. For a more precise investigation of how H5 contributes to DNA synthesis, we placed thets57 H5 allele in an otherwise wild-type viral background and also performed small interfering RNA-mediated depletion of H5. Finally, we generated a complementing cell line, CV-1–H5, which allowed us to generate a viral recombinant in which the H5 open reading frame was deleted and replaced with mCherry (vΔH5). Analysis of vΔH5 allowed us to demonstrate conclusively that viral DNA replication is abrogated in the absence of H5. The loss of H5 does not compromise the accumulation of other early viral replication proteins or the uncoating of the virion core, suggesting that H5 plays a direct and essential role in facilitating DNA synthesis.IMPORTANCEVariola virus, the causative agent of smallpox, is the most notorious member of thePoxviridaefamily. Poxviruses are unique among DNA viruses that infect mammalian cells, in that their replication is restricted to the cytoplasm of the cell. This physical autonomy from the nucleus has both cell biological and genetic ramifications. Poxviruses must establish cytoplasmic niches that support replication, and the genomes must encode the repertoire of proteins necessary for genome synthesis. Here we focus on H5, a multifunctional and abundant viral protein. We confirm that H5 associates with the DNA polymerase holoenzyme and localizes to the sites of DNA synthesis. By generating an H5-expressing cell line, we were able to isolate a deletion virus that lacks the H5 gene and show definitively that genome synthesis does not occur in the absence of H5. These data support the hypothesis that H5 is a crucial participant in cytoplasmic poxvirus genome replication.

scholarly journals Nuclear Reorganization of Mammalian DNA Synthesis Prior to Cell Cycle Exit

2004 ◽  
Vol 24 (2) ◽  
pp. 595-607 ◽  
Author(s):  
David A. Barbie ◽  
Brian A. Kudlow ◽  
Richard Frock ◽  
Jiyong Zhao ◽  
Brett R. Johnson ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Mammalian Cells ◽  
Large Scale ◽  
Replicative Senescence ◽  
S Phase ◽  
Serum Starvation ◽  
Cell Cycle Exit ◽  
Replication Foci

ABSTRACT In primary mammalian cells, DNA replication initiates in a small number of perinucleolar, lamin A/C-associated foci. During S-phase progression in proliferating cells, replication foci distribute to hundreds of sites throughout the nucleus. In contrast, we find that the limited perinucleolar replication sites persist throughout S phase as cells prepare to exit the cell cycle in response to contact inhibition, serum starvation, or replicative senescence. Proteins known to be involved in DNA synthesis, such as PCNA and DNA polymerase δ, are concentrated in perinucleolar foci throughout S phase under these conditions. Moreover, chromosomal loci are redirected toward the nucleolus and overlap with the perinucleolar replication foci in cells poised to undergo cell cycle exit. These same loci remain in the periphery of the nucleus during replication under highly proliferative conditions. These results suggest that mammalian cells undergo a large-scale reorganization of chromatin during the rounds of DNA replication that precede cell cycle exit.

scholarly journals TEMPERATURE-SENSITIVE MUTANTS OF A CHINESE HAMSTER CELL LINE. I. SELECTION OF CLONES WITH DEFECTIVE MACROMOLECULAR BIOSYNTHESIS

Genetics ◽  
1975 ◽  
Vol 80 (3) ◽  
pp. 549-566
Author(s):  
Donald J Roufa ◽  
Susan J Reed
Keyword(s):  
Amino Acids ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Mammalian Cells ◽  
Protein Biosynthesis ◽  
Chinese Hamster ◽  
Selection Procedure ◽  
Brdu Incorporation ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature

ABSTRACT Temperature-sensitive clones have been selected from a mutagenized culture of Chinese hamster lung cells by a procedure involving bromodeoxy-uridine (BrdU) incorporation and irradiation with black light. The selection procedure used in these studies was adapted from methods developed by others to yield mutants that cease DNA replication within a short time after they are transferred to nonpermissive temperature. After mutagenesis with ethyl methanosulfonate ten clones survived the selection procedure. Three of the clones (mutants) were temperature-sensitive as measured by growth properties. Two mutants ceased DNA synthesis within six hours of being shifted to 39° and the third mutant continued to synthesize DNA at nonpermissive temperature at a reduced rate for at least 24 hours. Thus, all three mutants survived the selection procedure for understandable reasons, since each was unable to incorporate sufficient BrdU at 39° to lethally protosensitize its DNA during the standard exposure period. The two mutants that cease DNA synthesis at high temperature (clones 115-47 and 115-53) also stop incorporating radioactive amino acids and uridine within six hours at 39°. Their complex phenotype, i.e. defective DNA, RNA and protein biosynthesis, is reversible. When these mutants were returned to 33° after 8 hours at 39°, both resumed DNA synthesis immediately (< 1 hour). Reversal of defective DNA synthesis in both mutants was sensitive to drugs that inhibit protein biosynthesis specifically. Those same drugs, as well as toxic amino acids analogs, also effected a striking mutant phenocopy in wild-type cells. The phenocopy produced by amino acid analogs that are incorporated into mammalian proteins suggested that one or more proteins must be synthesized continuously to support mammalian cells engaged in programmed DNA replication.

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