scholarly journals Aphidicolin does not inhibit DNA repair synthesis in ultraviolet-irradiated HeLa cells. A radioautographic study

1981 ◽  
Vol 199 (2) ◽  
pp. 453-455 ◽  
Author(s):  
N Hardt ◽  
G Pedrali-Noy ◽  
F Focher ◽  
S Spadari
Keyword(s):  
Dna Repair ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Hela Cells ◽  
Nuclear Dna ◽  
Detectable Effect ◽  
Repair Synthesis ◽  
Dna Polymerase Alpha ◽  
Dna Repair Synthesis

A radioautographic examination of nuclear DNA synthesis in unirradiated and u.v.-irradiated HeLa cells, in the presence and in the absence of aphidicolin, showed that aphidicolin inhibits nuclear DNA replication and has no detectable effect on DNA repair synthesis. Although the results establish that in u.v.-irradiated HeLa cells most of the DNA repair synthesis is not due to DNA polymerase alpha, they do not preclude a significant role for this enzyme in DNA repair processes.

scholarly journals Dynamics of protein binding to sites of nascent unscheduled DNA repair synthesis in non-proliferating cells

2020 ◽  
Author(s):  
Claudia Scalera ◽  
Ilaria Dutto ◽  
Francesca Barbazza ◽  
Raefa Abou Khouzam ◽  
Giulio Ticli ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Click Reaction ◽  
Excision Repair ◽  
Proliferating Cells ◽  
Repair Synthesis ◽  
Dynamic Association ◽  
Dna Repair Synthesis

ABSTRACTThe analysis of DNA repair mechanisms is of fundamental importance to understand how cells remove DNA damage and maintain their genome stability. Investigating the dynamic association of proteins at sites of active DNA synthesis has been successfully performed at DNA replication forks, providing important information on the process, and allowing the identification of new players acting at these sites. However, the applicability of these studies to DNA repair events at sites of nascent unscheduled DNA synthesis (UDS) in non-proliferating cells has been never tested. Here, we describe the analysis of dynamics association of protein participating in nucleotide excision repair (NER), and in other DNA repair processes, at sites of nascent UDS in non-proliferating cells, to avoid interference by DNA replication. Labeling with 5-ethynyl-2’-deoxyuridine (EdU) after DNA damage, followed by click reaction to biotinylate these sites, permits the analysis of dynamic association of proteins, such as DNA polymerases δ and κ, as well as PCNA, to active DNA repair synthesis sites. The suitability of this technique to identify new factors present at active UDS sites is illustrated by two examples of proteins previously unknown to participate in the UV-induced DNA repair process.

scholarly journals Involvement of deoxyribonucleic acid polymerase β in nuclear deoxyribonucleic acid synthesis

1978 ◽  
Vol 173 (1) ◽  
pp. 309-314 ◽  
Author(s):  
T R Butt ◽  
W M Wood ◽  
E L McKay ◽  
R L P Adams
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Deoxyribonucleic Acid ◽  
Nuclear Dna ◽  
Dna Polymerase Beta ◽  
Cell Nuclei ◽  
High Molecular Weight Dna ◽  
Dna Polymerase Alpha ◽  
Polymerase Beta

The effects on DNA synthesis in vitro in mouse L929-cell nuclei of differential extraction of DNA polymerases alpha and beta were studied. Removal of all measurable DNA polymerase alpha and 20% of DNA polymerase beta leads to a 40% fall in the replicative DNA synthesis. Removal of 70% of DNA polymerase beta inhibits replicative synthesis by 80%. In all cases the nuclear DNA synthesis is sensitive to N-ethylmaleimide and aCTP (arabinosylcytosine triphosphate), though less so than DNA polymerase alpha. Addition of deoxyribonuclease I to the nuclear incubation leads to synthesis of high-molecular-weight DNA in a repair reaction. This occurs equally in nuclei from non-growing or S-phase cells. The former nuclei lack DNA polymerase alpha and the reaction reflects the sensitivity of DNA polymerase beta to inhibiton by N-ethylmaleimide and aCTP.

In situ enzymology of DNA replication and ultraviolet-induced DNA repair synthesis in permeable human cells

Biochemistry ◽  
1988 ◽  
Vol 27 (19) ◽  
pp. 7247-7254 ◽  
Author(s):  
Steven L. Dresler ◽  
Mark G. Frattini ◽  
Rona M. Robinson-Hill
Keyword(s):  
Dna Repair ◽  
Dna Replication ◽  
Human Cells ◽  
Repair Synthesis ◽  
Dna Repair Synthesis

scholarly journals Initiation of simian virus 40 DNA replication in vitro: aphidicolin causes accumulation of early-replicating intermediates and allows determination of the initial direction of DNA synthesis.

1986 ◽  
Vol 6 (11) ◽  
pp. 3815-3825 ◽  
Author(s):  
R S Decker ◽  
M Yamaguchi ◽  
R Possenti ◽  
M L DePamphilis
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Early Gene ◽  
Initial Direction ◽  
Dna Polymerase Alpha

Aphidicolin, a specific inhibitor of DNA polymerase alpha, provided a novel method for distinguishing between initiation of DNA synthesis at the simian virus 40 (SV40) origin of replication (ori) and continuation of replication beyond ori. In the presence of sufficient aphidicolin to inhibit total DNA synthesis by 50%, initiation of DNA replication in SV40 chromosomes or ori-containing plasmids continued in vitro, whereas DNA synthesis in the bulk of SV40 replicative intermediate DNA (RI) that had initiated replication in vivo was rapidly inhibited. This resulted in accumulation of early RI in which most nascent DNA was localized within a 600- to 700-base-pair region centered at ori. Accumulation of early RI was observed only under conditions that permitted initiation of SV40 ori-dependent, T-antigen-dependent DNA replication and only when aphidicolin was added to the in vitro system. Increasing aphidicolin concentrations revealed that DNA synthesis in the ori region was not completely resistant to aphidicolin but simply less sensitive than DNA synthesis at forks that were farther away. Since DNA synthesized in the presence of aphidicolin was concentrated in the 300 base pairs on the early gene side of ori, we conclude that the initial direction of DNA synthesis was the same as that of early mRNA synthesis, consistent with the model proposed by Hay and DePamphilis (Cell 28:767-779, 1982). The data were also consistent with initiation of the first DNA chains in ori by CV-1 cell DNA primase-DNA polymerase alpha. Synthesis of pppA/G(pN)6-8(pdN)21-23 chains on a single-stranded DNA template by a purified preparation of this enzyme was completely resistant to aphidicolin, and further incorporation of deoxynucleotide monophosphates was inhibited. Therefore, in the presence of aphidicolin, this enzyme could initiate RNA-primed DNA synthesis at ori first in the early gene direction and then in the late gene direction, but could not continue DNA synthesis for an extended distance.

scholarly journals Induction of unscheduled DNA synthesis in human bone marrow cells by bifunctional alkylating agents

Blood ◽  
1979 ◽  
Vol 54 (6) ◽  
pp. 1320-1329
Author(s):  
R Lewensohn ◽  
U Ringborg
Keyword(s):  
Bone Marrow ◽  
Dna Repair ◽  
Dna Synthesis ◽  
Bone Marrow Cells ◽  
Alkylating Agents ◽  
Human Bone ◽  
Unscheduled Dna Synthesis ◽  
Repair Synthesis ◽  
Dna Repair Synthesis ◽  
Marrow Cells

A technique has been developed for handling human bone marrow cells intended for the examination of DNA repair synthesis. DNA-repair synthesis, induced by melphalan and nitrogen mustard, was measured as the incorporation of 3H-thymidine, registered by autoradiography as unscheduled DNA synthesis (UDS). Comparison of various cell populations disclosed considerable differences in their UDS level, this generally being greatest for the blast populations. During maturation of both myelopoietic and erythropoietic cells, there was a decrease in the UDS level, which was lowest for the end-cell stage. The lymphocytes and monocytes differed considerably in their capacity for UDS. The developed technique would appear to offer an opportunity for determining the capacity for DNA-repair synthesis in malignant bone marrow cells, a factor that has been linked to sensitivity to alkylating agents.

scholarly journals The Base Excision Repair Pathway in the Nematode Caenorhabditis elegans

2020 ◽  
Vol 8 ◽  
Author(s):  
Noha Elsakrmy ◽  
Qiu-Mei Zhang-Akiyama ◽  
Dindial Ramotar
Keyword(s):  
Dna Repair ◽  
Caenorhabditis Elegans ◽  
Dna Polymerase ◽  
Base Excision Repair ◽  
Excision Repair ◽  
Dna Glycosylases ◽  
Repair Synthesis ◽  
C Elegans ◽  
Base Excision ◽  
Dna Repair Synthesis

Exogenous and endogenous damage to the DNA is inevitable. Several DNA repair pathways including base excision, nucleotide excision, mismatch, homologous and non-homologous recombinations are conserved across all organisms to faithfully maintain the integrity of the genome. The base excision repair (BER) pathway functions to repair single-base DNA lesions and during the process creates the premutagenic apurinic/apyrimidinic (AP) sites. In this review, we discuss the components of the BER pathway in the nematode Caenorhabditis elegans and delineate the different phenotypes caused by the deletion or the knockdown of the respective DNA repair gene, as well as the implications. To date, two DNA glycosylases have been identified in C. elegans, the monofunctional uracil DNA glycosylase-1 (UNG-1) and the bifunctional endonuclease III-1 (NTH-1) with associated AP lyase activity. In addition, the animal possesses two AP endonucleases belonging to the exonuclease-3 and endonuclease IV families and in C. elegans these enzymes are called EXO-3 and APN-1, respectively. In mammalian cells, the DNA polymerase, Pol beta, that is required to reinsert the correct bases for DNA repair synthesis is not found in the genome of C. elegans and the evidence indicates that this role could be substituted by DNA polymerase theta (POLQ), which is known to perform a function in the microhomology-mediated end-joining pathway in human cells. The phenotypes observed by the C. elegans mutant strains of the BER pathway raised many challenging questions including the possibility that the DNA glycosylases may have broader functional roles, as discuss in this review.

scholarly journals DNA repair synthesis during base excision repair in vitro is catalyzed by DNA polymerase epsilon and is influenced by DNA polymerases alpha and delta in Saccharomyces cerevisiae.

1993 ◽  
Vol 13 (2) ◽  
pp. 1051-1058 ◽  
Author(s):  
Z Wang ◽  
X Wu ◽  
E C Friedberg
Keyword(s):  
Dna Repair ◽  
Dna Polymerase ◽  
Base Excision Repair ◽  
Excision Repair ◽  
Repair Synthesis ◽  
Dna Polymerase Epsilon ◽  
Nuclear Extracts ◽  
Polymerase Epsilon ◽  
Base Excision ◽  
Dna Repair Synthesis

Base excision repair is an important mechanism for correcting DNA damage produced by many physical and chemical agents. We have examined the effects of the REV3 gene and the DNA polymerase genes POL1, POL2, and POL3 of Saccharomyces cerevisiae on DNA repair synthesis is nuclear extracts. Deletional inactivation of REV3 did not affect repair synthesis in the base excision repair pathway. Repair synthesis in nuclear extracts of pol1, pol2, and pol3 temperature-sensitive mutants was normal at permissive temperatures. However, repair synthesis in pol2 nuclear extracts was defective at the restrictive temperature of 37 degrees C and could be complemented by the addition of purified yeast DNA polymerase epsilon. Repair synthesis in pol1 nuclear extracts was proficient at the restrictive temperature unless DNA polymerase alpha was inactivated prior to the initiation of DNA repair. Thermal inactivation of DNA polymerase delta in pol3 nuclear extracts enhanced DNA repair synthesis approximately 2-fold, an effect which could be specifically reversed by the addition of purified yeast DNA polymerase delta to the extract. These results demonstrate that DNA repair synthesis in the yeast base excision repair pathway is catalyzed by DNA polymerase epsilon but is apparently modulated by the presence of DNA polymerases alpha and delta.

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