scholarly journals Repair of abasic sites by mammalian cell extracts

1994 ◽  
Vol 304 (3) ◽  
pp. 699-705 ◽  
Author(s):  
G Frosina ◽  
P Fortini ◽  
O Rossi ◽  
F Carrozzino ◽  
A Abbondandolo ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Excision Repair ◽  
Chinese Hamster ◽  
Pyrimidine Dimer ◽  
Repair Synthesis ◽  
Repair Patch ◽  
Cell Extracts ◽  
Pyrimidine Dimers ◽  
Ap Sites ◽  
Ap Site

Hamster cell extracts that perform repair synthesis on covalently closed circular DNA containing pyrimidine dimers, were used to study the repair of apurinic/apyrimidinic (AP) sites and methoxyamine (MX)-modified AP sites. Plasmid molecules were heat-treated at pH 5 and incubated with MX when required. The amount of damage introduced ranged from 0.2 to 0.9 AP sites/kb. Extracts were prepared from the Chinese hamster ovary CHO-9 cell line and from its derivative, 43-3B clone which is mutated in the nucleotide excision repair (NER) ERCC1 gene. AP and MX-AP sites stimulated repair synthesis by CHO-9 cell extracts. The level of synthesis correlated with the number of lesions and was of similar magnitude to the repair stimulated by 4.3 u.v. photoproducts/kb. Repair of AP and MX-AP sites was faster than the repair of u.v. damage and was independent of ERCC1 gene product. The high level of repair replication was due to a very efficient and rapid incision of plasmids carrying AP or MX-AP sites, performed by abundant AP endonucleases present in the extract. The calculated average repair patch sizes were: 7 nucleotides per AP site; 10 nucleotides per MX-AP site; 28 nucleotides per (6-4) u.v. photoproduct or cyclobutane pyrimidine dimer. The data indicate that AP and MX-AP sites are very efficiently repaired by base-excision repair in mammalian cells and suggest that MX-AP sites may also be processed via alternative repair mechanisms.

scholarly journals Cytotoxicity and Mutagenicity of Narrowband UVB to Mammalian Cells

Genes ◽  
2020 ◽  
Vol 11 (6) ◽  
pp. 646
Author(s):  
Dylan J. Buglewicz ◽  
Jacob T. Mussallem ◽  
Alexis H. Haskins ◽  
Cathy Su ◽  
Junko Maeda ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Skin Diseases ◽  
Excision Repair ◽  
Chinese Hamster ◽  
Ultraviolet B ◽  
Pyrimidine Dimer ◽  
Ros Generation ◽  
Narrowband Uvb ◽  
The Difference ◽  
Uvb Exposure

Phototherapy using narrowband ultraviolet-B (NB-UVB) has been shown to be more effective than conventional broadband UVB (BB-UVB) in treating a variety of skin diseases. To assess the difference in carcinogenic potential between NB-UVB and BB-UVB, we investigated the cytotoxicity via colony formation assay, genotoxicity via sister chromatid exchange (SCE) assay, mutagenicity via hypoxanthine phosphoribosyltransferase (HPRT) mutation assay, as well as cyclobutane pyrimidine dimer (CPD) formation and reactive oxygen species (ROS) generation in Chinese hamster ovary (CHO) and their NER mutant cells. The radiation dose required to reduce survival to 10% (D10 value) demonstrated BB-UVB was 10 times more cytotoxic than NB-UVB, and revealed that NB-UVB also induces DNA damage repaired by nucleotide excision repair. We also found that BB-UVB more efficiently induced SCEs and HPRT mutations per absorbed energy dosage (J/m2) than NB-UVB. However, SCE and HPRT mutation frequencies were observed to rise in noncytotoxic dosages of NB-UVB exposure. BB-UVB and NB-UVB both produced a significant increase in CPD formation and ROS formation (p < 0.05); however, higher dosages were required for NB-UVB. These results suggest that NB-UVB is less cytotoxic and genotoxic than BB-UVB, but can still produce genotoxic effects even at noncytotoxic doses.

Preferential repair of damage in actively transcribed DNA sequences in vivo

Genome ◽  
1989 ◽  
Vol 31 (2) ◽  
pp. 605-611 ◽  
Author(s):  
Philip C. Hanawalt
Keyword(s):  
Dna Repair ◽  
Dna Sequences ◽  
Mammalian Cells ◽  
Excision Repair ◽  
Chinese Hamster ◽  
Human Cells ◽  
Dhfr Gene ◽  
Pyrimidine Dimers ◽  
Cross Links

My colleagues and I have discovered intragenomic heterogeneity in DNA repair in mammalian cells. Consequences of unrepaired DNA damage depend upon the precise location of the damage with respect to relevant genes. It is therefore important to understand rules governing accessibility of specific DNA sequences in chromatin to damage and repair. The efficiency of removal of pyrimidine dimers has been determined in the active dihydrofolate reductase (DHFR) gene in Chinese hamster ovary (CHO) cells. Repair within the gene was shown to be much more efficient than that in nontranscribed downstream sequences or in the genome overall. Preferential repair of active and essential genes such as DHFR may account for the fact that rodent cells are as uv-resistant as human cells in spite of their much lower overall repair efficiencies. In repair-proficient human cells the rate of repair in the DHFR gene is greater than that in the overall genome or in nontranscribed α-DNA sequences. The efficiency of removal of pyrimidine dimers is much higher in the transcribed than the nontranscribed DNA strands of the DHFR gene in both CHO and human cells. An excision–repair complex may be directly coupled to the transcription machinery to ensure early removal of transcription-blocking lesions in active genes. Sequences in the active c-abl proto-oncogene are repaired much more efficiently than are sequences containing the inactive c-mos proto-oncogene in Swiss mouse 3T3 cells. Tissue-specific and cell-specific differences in the coordinate regulation of proto-oncogene expression and DNA repair may account for corresponding differences in the carcinogenic response. Efficient replicative bypass of persisting psoralen monoadducts, but not interstrand cross-links, was demonstrated in the human DHFR gene. It is likely that most bulky lesions in mammalian DNA, other than cross-links, do not pose insurmountable problems for replication in vivo, but they must be removed from essential transcribed sequences to maintain cellular viability.Key words: DNA repair, chromatin, transcription, mammalian cells, pyrimidine dimers, ultraviolet light, DNA cross-links.

scholarly journals Abasic Sites in the Transcribed Strand of Yeast DNA Are Removed by Transcription-Coupled Nucleotide Excision Repair

2010 ◽  
Vol 30 (13) ◽  
pp. 3206-3215 ◽  
Author(s):  
Nayun Kim ◽  
Sue Jinks-Robertson
Keyword(s):  
Nucleotide Excision Repair ◽  
Excision Repair ◽  
Genome Integrity ◽  
Genetic Studies ◽  
Abasic Sites ◽  
Dna And Rna ◽  
Nucleotide Excision ◽  
Ap Sites ◽  
Base Excision ◽  
Ap Site

ABSTRACT Abasic (AP) sites are potent blocks to DNA and RNA polymerases, and their repair is essential for maintaining genome integrity. Although AP sites are efficiently dealt with through the base excision repair (BER) pathway, genetic studies suggest that repair also can occur via nucleotide excision repair (NER). The involvement of NER in AP-site removal has been puzzling, however, as this pathway is thought to target only bulky lesions. Here, we examine the repair of AP sites generated when uracil is removed from a highly transcribed gene in yeast. Because uracil is incorporated instead of thymine under these conditions, the position of the resulting AP site is known. Results demonstrate that only AP sites on the transcribed strand are efficient substrates for NER, suggesting the recruitment of the NER machinery by an AP-blocked RNA polymerase. Such transcription-coupled NER of AP sites may explain previously suggested links between the BER pathway and transcription.

scholarly journals An interaction between the mammalian DNA repair protein XRCC1 and DNA ligase III.

1994 ◽  
Vol 14 (1) ◽  
pp. 68-76 ◽  
Author(s):  
K W Caldecott ◽  
C K McKeown ◽  
J D Tucker ◽  
S Ljungquist ◽  
L H Thompson
Keyword(s):  
Dna Repair ◽  
Affinity Chromatography ◽  
Mammalian Cells ◽  
Excision Repair ◽  
Affinity Purification ◽  
Alkylating Agents ◽  
Chinese Hamster ◽  
Dna Ligase ◽  
Base Excision ◽  
Dna Ligase Iii

XRCC1, the human gene that fully corrects the Chinese hamster ovary DNA repair mutant EM9, encodes a protein involved in the rejoining of DNA single-strand breaks that arise following treatment with alkylating agents or ionizing radiation. In this study, a cDNA minigene encoding oligohistidine-tagged XRCC1 was constructed to facilitate affinity purification of the recombinant protein. This construct, designated pcD2EHX, fully corrected the EM9 phenotype of high sister chromatid exchange, indicating that the histidine tag was not detrimental to XRCC1 activity. Affinity chromatography of extract from EM9 cells transfected with pcD2EHX resulted in the copurification of histidine-tagged XRCC1 and DNA ligase III activity. Neither XRCC1 or DNA ligase III activity was purified during affinity chromatography of extract from EM9 cells transfected with pcD2EX, a cDNA minigene that encodes untagged XRCC1, or extract from wild-type AA8 or untransfected EM9 cells. The copurification of DNA ligase III activity with histidine-tagged XRCC1 suggests that the two proteins are present in the cell as a complex. Furthermore, DNA ligase III activity was present at lower levels in EM9 cells than in AA8 cells and was returned to normal levels in EM9 cells transfected with pcD2EHX or pcD2EX. These findings indicate that XRCC1 is required for normal levels of DNA ligase III activity, and they implicate a major role for this DNA ligase in DNA base excision repair in mammalian cells.

scholarly journals Photolyases from Saccharomyces cerevisiae and Escherichia coli recognize common binding determinants in DNA containing pyrimidine dimers.

1989 ◽  
Vol 9 (11) ◽  
pp. 4777-4788 ◽  
Author(s):  
M Baer ◽  
G B Sancar
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Nucleotide Excision Repair ◽  
Excision Repair ◽  
Spectroscopic Studies ◽  
Pyrimidine Dimer ◽  
Nucleotide Sequence Analysis ◽  
Binding Motif ◽  
Pyrimidine Dimers ◽  
Nucleotide Excision

DNA photolyases catalyze the light-dependent repair of pyrimidine dimers in DNA. The results of nucleotide sequence analysis and spectroscopic studies demonstrated that photolyases from Saccharomyces cerevisiae and Escherichia coli share 37% amino acid sequence homology and contain identical chromophores. Do the similarities between these two enzymes extend to their interactions with DNA containing pyrimidine dimers, or does the organization of DNA into nucleosomes in S. cerevisiae necessitate alternative or additional recognition determinants? To answer this question, we used chemical and enzymatic techniques to identify the contacts made on DNA by S. cerevisiae photolyase when it is bound to a pyrimidine dimer and compared these contacts with those made by E. coli photolyase and by a truncated derivative of the yeast enzyme when bound to the same substrate. We found evidence for a common set of interactions between the photolyases and specific phosphates in the backbones of both strands as well as for interactions with bases in both the major and minor grooves of dimer-containing DNA. Superimposed on this common pattern were significant differences in the contributions of specific contacts to the overall binding energy, in the interactions of the enzymes with groups on the complementary strand, and in the extent to which other DNA-binding proteins were excluded from the region around the dimer. These results provide strong evidence both for a conserved dimer-binding motif and for the evolution of new interactions that permit photolyases to also act as accessory proteins in nucleotide excision repair. The locations of the specific contacts made by the yeast enzyme indicate that the mechanism of nucleotide excision repair in this organism involves incision(s) at a distance from the pyrimidine dimer.

Xeroderma pigmentosum complementation group C cells remove pyrimidine dimers selectively from the transcribed strand of active genes

1991 ◽  
Vol 11 (8) ◽  
pp. 4128-4134
Author(s):  
J Venema ◽  
A van Hoffen ◽  
V Karcagi ◽  
A T Natarajan ◽  
A A van Zeeland ◽  
...  
Keyword(s):  
Xeroderma Pigmentosum ◽  
Mammalian Cells ◽  
Excision Repair ◽  
Complementation Group ◽  
C Cells ◽  
Dhfr Gene ◽  
Normal Cells ◽  
Pyrimidine Dimers ◽  
Normal Human ◽  
Active Genes

We have measured the removal of UV-induced pyrimidine dimers from DNA fragments of the adenosine deaminase (ADA) and dihydrofolate reductase (DHFR) genes in primary normal human and xeroderma pigmentosum complementation group C (XP-C) cells. Using strand-specific probes, we show that in normal cells, preferential repair of the 5' part of the ADA gene is due to the rapid and efficient repair of the transcribed strand. Within 8 h after irradiation with UV at 10 J m-2, 70% of the pyrimidine dimers in this strand are removed. The nontranscribed strand is repaired at a much slower rate, with 30% dimers removed after 8 h. Repair of the transcribed strand in XP-C cells occurs at a rate indistinguishable from that in normal cells, but the nontranscribed strand is not repaired significantly in these cells. Similar results were obtained for the DHFR gene. In the 3' part of the ADA gene, however, both normal and XP-C cells perform fast and efficient repair of either strand, which is likely to be caused by the presence of transcription units on both strands. The factor defective in XP-C cells is apparently involved in the processing of DNA damage in inactive parts of the genome, including nontranscribed strands of active genes. These findings have important implications for the understanding of the mechanism of UV-induced excision repair and mutagenesis in mammalian cells.

The RAD7 and RAD16 genes, which are essential for pyrimidine dimer removal from the silent mating type loci, are also required for repair of the nontranscribed strand of an active gene in Saccharomyces cerevisiae

1994 ◽  
Vol 14 (9) ◽  
pp. 6135-6142
Author(s):  
R Verhage ◽  
A M Zeeman ◽  
N de Groot ◽  
F Gleig ◽  
D D Bang ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mating Type ◽  
Excision Repair ◽  
Complementation Group ◽  
Pyrimidine Dimer ◽  
Gene Products ◽  
Pyrimidine Dimers ◽  
Nucleotide Excision ◽  
Active Genes ◽  
Silent Mating Type Loci

The rad16 mutant of Saccharomyces cerevisiae was previously shown to be impaired in removal of UV-induced pyrimidine dimers from the silent mating-type loci (D. D. Bang, R. A. Verhage, N. Goosen, J. Brouwer, and P. van de Putte, Nucleic Acids Res. 20:3925-3931, 1992). Here we show that rad7 as well as rad7 rad16 double mutants have the same repair phenotype, indicating that the RAD7 and RAD16 gene products might operate in the same nucleotide excision repair subpathway. Dimer removal from the genome overall is essentially incomplete in these mutants, leaving about 20 to 30% of the DNA unrepaired. Repair analysis of the transcribed RPB2 gene shows that the nontranscribed strand is not repaired at all in rad7 and rad16 mutants, whereas the transcribed strand is repaired in these mutants at a fast rate similar to that in RAD+ cells. When the results obtained with the RPB2 gene can be generalized, the RAD7 and RAD16 proteins not only are essential for repair of silenced regions but also function in repair of nontranscribed strands of active genes in S. cerevisiae. The phenotype of rad7 and rad16 mutants closely resembles that of human xeroderma pigmentosum complementation group C (XP-C) cells, suggesting that RAD7 and RAD16 in S. cerevisiae function in the same pathway as the XPC gene in human cells. RAD4, which on the basis of sequence homology has been proposed to be the yeast XPC counterpart, seems to be involved in repair of both inactive and active yeast DNA, challenging the hypothesis that RAD4 and XPC are functional homologs.

scholarly journals Generation of single-nucleotide repair patches following excision of uracil residues from DNA.

1992 ◽  
Vol 12 (4) ◽  
pp. 1605-1612 ◽  
Author(s):  
G Dianov ◽  
A Price ◽  
T Lindahl
Keyword(s):  
Mammalian Cell ◽  
Excision Repair ◽  
Enzymatic Digestion ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Repair Synthesis ◽  
Single Nucleotide ◽  
Cell Extracts ◽  
Dna Repair Synthesis

The extent and location of DNA repair synthesis in a double-stranded oligonucleotide containing a single dUMP residue have been determined. Gently prepared Escherichia coli and mammalian cell extracts were employed for excision repair in vitro. The size of the resynthesized patch was estimated by restriction enzyme analysis of the repaired oligonucleotide. Following enzymatic digestion and denaturing gel electrophoresis, the extent of incorporation of radioactively labeled nucleotides in the vicinity of the lesion was determined by autoradiography. Cell extracts of E. coli and of human cell lines were shown to carry out repair mainly by replacing a single nucleotide. No significant repair replication on the 5' side of the lesion was observed. The data indicate that, after cleavage of the dUMP residue by uracil-DNA glycosylase and incision of the resultant apurinic-apyrimidinic site by an apurinic-apyrimidinic endonuclease activity, the excision step is catalyzed usually by a DNA deoxyribophosphodiesterase rather than by an exonuclease. Gap-filling and ligation complete the repair reaction. Experiments with enzyme inhibitors in mammalian cell extracts suggest that the repair replication step is catalyzed by DNA polymerase beta.

scholarly journals Xeroderma pigmentosum complementation group C cells remove pyrimidine dimers selectively from the transcribed strand of active genes.

1991 ◽  
Vol 11 (8) ◽  
pp. 4128-4134 ◽  
Author(s):  
J Venema ◽  
A van Hoffen ◽  
V Karcagi ◽  
A T Natarajan ◽  
A A van Zeeland ◽  
...  
Keyword(s):  
Xeroderma Pigmentosum ◽  
Mammalian Cells ◽  
Excision Repair ◽  
Complementation Group ◽  
C Cells ◽  
Dhfr Gene ◽  
Normal Cells ◽  
Pyrimidine Dimers ◽  
Normal Human ◽  
Active Genes

We have measured the removal of UV-induced pyrimidine dimers from DNA fragments of the adenosine deaminase (ADA) and dihydrofolate reductase (DHFR) genes in primary normal human and xeroderma pigmentosum complementation group C (XP-C) cells. Using strand-specific probes, we show that in normal cells, preferential repair of the 5' part of the ADA gene is due to the rapid and efficient repair of the transcribed strand. Within 8 h after irradiation with UV at 10 J m-2, 70% of the pyrimidine dimers in this strand are removed. The nontranscribed strand is repaired at a much slower rate, with 30% dimers removed after 8 h. Repair of the transcribed strand in XP-C cells occurs at a rate indistinguishable from that in normal cells, but the nontranscribed strand is not repaired significantly in these cells. Similar results were obtained for the DHFR gene. In the 3' part of the ADA gene, however, both normal and XP-C cells perform fast and efficient repair of either strand, which is likely to be caused by the presence of transcription units on both strands. The factor defective in XP-C cells is apparently involved in the processing of DNA damage in inactive parts of the genome, including nontranscribed strands of active genes. These findings have important implications for the understanding of the mechanism of UV-induced excision repair and mutagenesis in mammalian cells.

scholarly journals Bacteriophage-T4 and Micrococcus luteus UV endonucleases are not endonucleases but β-elimination and sometimes βδ-elimination catalysts

1989 ◽  
Vol 259 (3) ◽  
pp. 751-759 ◽  
Author(s):  
V Bailly ◽  
B Sente ◽  
W G Verly
Keyword(s):  
Enzyme Preparation ◽  
Micrococcus Luteus ◽  
Bacteriophage T4 ◽  
Pyrimidine Dimer ◽  
Elimination Reaction ◽  
Ap Sites ◽  
Beta Elimination ◽  
Ap Site ◽  
Polynucleotide Chain

Bacteriophage-T4 UV endonuclease nicks the C(3′)-O-P bond 3′ to AP (apurinic or apyrimidinic) sites by a beta-elimination reaction. The breakage of this bond is sometimes followed by the nicking of the C(5′)-O-P bond 5′ to the AP site, leaving a 3′-phosphate end; delta-elimination is proposed as a mechanism to explain this second reaction. The AP site formed when this enzyme acts on a pyrimidine dimer in a polynucleotide chain undergoes the same nicking reactions. Micrococcus luteus UV endonuclease also nicks the C(3′)-O-P bond 3′ to AP sites by a beta-elimination reaction. No subsequent delta-elimination was observed, but this might be due to the presence of 2-mercaptoethanol in the enzyme preparation.

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