scholarly journals Repair of depurinated DNA with enzymes from rat liver chromatin

1984 ◽  
Vol 220 (1) ◽  
pp. 133-137 ◽  
Author(s):  
C Goffin ◽  
W G Verly
Keyword(s):  
Rat Liver ◽  
Nuclear Dna ◽  
Eukaryotic Cell ◽  
Dna Ligase ◽  
Polymerase Beta ◽  
Liver Chromatin ◽  
Dna Strand ◽  
T7 Phage ◽  
Ap Site

DNA from T7 phage containing AP (apurinic/apyrimidinic) sites was repaired by the successive actions of three chromatin enzymes [AP endodeoxyribonuclease, DNAase IV (5′----3′-exodeoxyribonuclease) and DNA polymerase-beta] prepared from rat liver and T4-phage DNA ligase. Since DNA ligase is also found in rat liver chromatin, all the activities used for the successful repair in vitro are thus present in the chromatin of a eukaryotic cell. Our results show, in particular, that the chromatin DNAase IV is capable of excising the AP site from the DNA strand nicked by the chromatin AP endodeoxyribonuclease. We did not try to combine all the enzymes, since competition between some of them might have prevented the repair; we have, for instance, shown that DNA ligase can seal the incision 5′ to the AP site made by the AP endodeoxyribonuclease. Changes in chromatin structure during repair might perhaps prevent this competition when nuclear DNA is repaired in the living cell.

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.

scholarly journals Characterization of double-stranded ribonucleic acid sequences present in the initial transcription products of rat liver chromatin

1977 ◽  
Vol 165 (2) ◽  
pp. 237-245 ◽  
Author(s):  
E Pays
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Rat Liver ◽  
Rna Sequences ◽  
Complementary Sequences ◽  
Exogenous Rna ◽  
Liver Chromatin ◽  
Low Ionic Strength

At low ionic strength and with a low exogenous RNA polymerase/DNA ratio, rat liver chromatin directs the synthesis in vitro of RNA sequences rich in double-stranded segments. All the transcripts contain at least one double-stranded sequence. Most of the double-stranded segments are formed by intramolecular base-pairing of inverted complementary sequences separated by a single-stranded loop. They are heterogeneous in size, 35-45% of them being more than 80 nucleotides long. They contain 61-64% G+C, whether synthesized by rat liver RNA polymerase (form B) or Escherichia coli RNA polymerase. The largest double-stranded sequences are found in the largest transcripts, and are the most thermostable. The fidelity of base-matching is better in double-stranded transcripts synthesized on rat liver chromatin by homologous polymerase than in those synthesized on it by a bacterial polymerase, or in those synthesized by either of the two polymerases on pure DNA.

scholarly journals Differential inhibition of rat liver DNA polymerases in vitro by direct-acting carcinogens and the protective effect of a thiol reducing agent

1981 ◽  
Vol 193 (3) ◽  
pp. 985-990 ◽  
Author(s):  
J Y Chan ◽  
F F Becker
Keyword(s):  
Dna Polymerase ◽  
Rat Liver ◽  
Reducing Agent ◽  
Gamma Activity ◽  
Repair Enzyme ◽  
Dna Polymerase Alpha ◽  
Polymerase Beta ◽  
Direct Acting

The direct-acting carcinogens acetoxyacetylaminofluorene, methylnitrosourea, and N-methyl-N'-nitro-N-nitrosoguanidine were tested for their ability to inhibit rat liver DNA polymerase-alpha, -beta, and -gamma activity in vitro. DNA polymerase-alpha was the most sensitive, polymerase-beta was the most resistant, and polymerase-gamma exhibited an intermediate response. When the reactions were reassayed in the presence and absence of dithiothreitol, a thiol reducing agent, it was shown that the inhibition by carcinogens was generally reversible with increasing dithiothreitol, except that polymerase-beta recovered only 80-90% of control values. These and binding data suggest that DNA polymerase-beta, the putative repair enzyme, is highly resistant to carcinogen damage. This resistance may contribute to the retention of normal function and fidelity of the repair enzyme during carcinogen exposure in vivo and to a normal cellular repair.

scholarly journals DNA ligase I variants fail in the ligation of mutagenic repair intermediates with mismatches and oxidative DNA damage

Mutagenesis ◽  
2020 ◽  
Author(s):  
Qun Tang ◽  
Pradnya Kamble ◽  
Melike Çağlayan
Keyword(s):  
Dna Polymerase ◽  
Oxidative Dna Damage ◽  
Deficiency Syndrome ◽  
Dna Ligase ◽  
Synonymous Mutations ◽  
Dna Ligase I ◽  
Dna Replication And Repair ◽  
Dna Mismatches ◽  
Dna Strand

Abstract DNA ligase I (LIG1) joins DNA strand breaks during DNA replication and repair transactions and contributes to genome integrity. The mutations (P529L, E566K, R641L and R771W) in LIG1 gene are described in patients with LIG1-deficiency syndrome that exhibit immunodeficiency. LIG1 senses 3’-DNA ends with a mismatch or oxidative DNA base inserted by a repair DNA polymerase. However, the ligation efficiency of the LIG1 variants for DNA polymerase-promoted mutagenesis products with 3’-DNA mismatches or 8-oxo-2’-deoxyguanosine (8-oxodG) remains undefined. Here, we report that R641L and R771W fail in the ligation of nicked DNA with 3’-8-oxodG, leading to an accumulation of 5’-AMP-DNA intermediates in vitro. Moreover, we found that the presence of all possible 12 non-canonical base pairs variously impacts the ligation efficiency by P529L and R771W depending on the architecture at the DNA end, whereas E566K exhibits no activity against all substrates tested. Our results contribute to the understanding of the substrate specificity and mismatch discrimination of LIG1 for mutagenic repair intermediates and the effect of non-synonymous mutations on ligase fidelity.

scholarly journals Possible roles of β-elimination and δ-elimination reactions in the repair of DNA containing AP (apurinic/apyrimidinic) sites in mammalian cells

1988 ◽  
Vol 253 (2) ◽  
pp. 553-559 ◽  
Author(s):  
V Bailly ◽  
W G Verly
Keyword(s):  
Mammalian Cells ◽  
Free Sugar ◽  
Ap Endonuclease ◽  
Phosphodiester Bond ◽  
Polymerase Beta ◽  
Beta Elimination ◽  
Elimination Reactions ◽  
The One ◽  
Ap Site

Histones and polyamines nick the phosphodiester bond 3′ to AP (apurinic/apyrimidinic) sites in DNA by inducing a beta-elimination reaction, which can be followed by delta-elimination. These beta- and delta-elimination reactions might be important for the repair of AP sites in chromatin DNA in either of two ways. In one pathway, after the phosphodiester bond 5′ to the AP site has been hydrolysed with an AP endonuclease, the 5′-terminal base-free sugar 5′-phosphate is released by beta-elimination. The one-nucleotide gap limited by 3′-OH and 5′-phosphate ends is then closed by DNA polymerase-beta and DNA ligase. We have shown in vitro that such a repair is possible. In the other pathway, the nicking 3′ to the AP site by beta-elimination occurs first. We have shown that the 3′-terminal base-free sugar so produced cannot be released by the chromatin AP endonuclease from rat liver. But it can be released by delta-elimination, leaving a gap limited by 3′-phosphate and 5′-phosphate. After conversion of the 3′-phosphate into a 3′-OH group by the chromatin 3′-phosphatase, there will be the same one-nucleotide gap, limited by 3′-OH and 5′-phosphate, as that formed by the successive actions of the AP endonuclease and the beta-elimination catalyst in the first pathway.

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