scholarly journals Digestion of damaged DNA by the T7 DNA polymerase-exonuclease

1993 ◽  
Vol 293 (2) ◽  
pp. 451-453 ◽  
Author(s):  
D R Koehler ◽  
P C Hanawalt
Keyword(s):  
Dna Polymerase ◽  
Exonuclease Activity ◽  
Dna Fragments ◽  
Cyclobutane Pyrimidine Dimers ◽  
Reaction Conditions ◽  
Pyrimidine Dimers ◽  
Phage T7 ◽  
Damaged Dna

We have investigated the 3′-5′-exonuclease activity of phage T7 DNA polymerase for its usefulness as an approach for the detection of lesions in DNA. Unlike the T4 DNA polymerase-exonuclease, which is commonly used to map the position and frequency of lesions in very small DNA fragments, T7 DNA polymerase-exonuclease is able to hydrolyse almost completely the large fragments from KpnI-restricted mammalian DNA. However, we found that the exonuclease was also able to hydrolyse DNA containing several kinds of lesions: cyclobutane pyrimidine dimers, thymine glycols, and mono-adducts of 4′-hydroxymethyl-4,5′,8-trimethylpsoralen and 5′-methyl-isopsoralen. Modifications of the reaction conditions did not significantly alter the extent of hydrolysis. These properties distinguish the T7 DNA polymerase-exonuclease from the T4 DNA polymerase-exonuclease and make the T7 DNA polymerase-exonuclease unsuitable for detecting several types of lesions in DNA.

scholarly journals 2-Chloro-2′-deoxyadenosine monophosphate residues in DNA enhance susceptibility to 3′ → 5′ exonucleases

1994 ◽  
Vol 302 (2) ◽  
pp. 567-571 ◽  
Author(s):  
P Hentosh ◽  
P Grippo
Keyword(s):  
Dna Polymerase ◽  
Nucleoside Analogues ◽  
Duplex Dna ◽  
Exonuclease Activity ◽  
Klenow Fragment ◽  
Purine Nucleotide ◽  
Nucleotide Analogue ◽  
Pyrimidine Dimers ◽  
Deoxyadenosine Triphosphate ◽  
Dna Polymerase Ii

2-Chloro-2′-deoxyadenosine triphosphate, a purine nucleotide analogue and potent antileukaemic agent, was incorporated into double-stranded 36-mers in place of dATP to investigate the effects of 2-chloroadenine (ClAde) on DNA polymerase-associated 3′-->5′ exonuclease activity. ClAde residues within one strand of duplex DNA did not inhibit exonuclease activity; on the contrary, ClAde-containing minus strands were digested to a greater extent than was control DNA in the absence of deoxyribonucleoside triphosphates by Escherichia coli Klenow fragment, yeast DNA polymerase II and T4 DNA polymerase. After a 30 min incubation with 5 units of Klenow fragment, approximately 65% of control DNA remained in DNA fragments of 26 bases or larger compared with only approximately 25% of ClAde-substituted substrates. Unsubstituted plus strands opposite a ClAde-containing strand were likewise digested more quickly by 3′-->5′ exonuclease, but only in the vicinity of the ClAde sites. Approx. 63% of the plus strands from ClAde-containing oligomers were less than 24 bases in length after a 25 min digestion period with Klenow fragment compared with only approximately 32% of control DNA. Such results indicate that, unlike other base modifications such as pyrimidine dimers, methoxy psoralen adducts and certain nucleoside analogues, all of which inhibit or decrease the rate of strand degradation by 3′-->5′ exonucleases, incorporated ClAde enhances strand degradation of duplex DNA.

Cyclobutane pyrimidine dimers and (6-4) photoproducts block polymerization by DNA polymerase I

Biochemistry ◽  
1985 ◽  
Vol 24 (21) ◽  
pp. 5723-5728 ◽  
Author(s):  
Gerald L. Chan ◽  
Paul W. Doetsch ◽  
William A. Haseltine
Keyword(s):  
Dna Polymerase ◽  
Dna Polymerase I ◽  
Cyclobutane Pyrimidine Dimers ◽  
Pyrimidine Dimers ◽  
Polymerase I

scholarly journals Characterization of a Y-Family DNA Polymerase eta from the Eukaryotic ThermophileAlvinella pompejana

2010 ◽  
Vol 2010 ◽  
pp. 1-13 ◽  
Author(s):  
Sayo Kashiwagi ◽  
Isao Kuraoka ◽  
Yoshie Fujiwara ◽  
Kenichi Hitomi ◽  
Quen J. Cheng ◽  
...  
Keyword(s):  
Organic Solvents ◽  
Dna Polymerase ◽  
Nuclear Antigen ◽  
Cyclobutane Pyrimidine Dimers ◽  
Thymine Glycol ◽  
Polymerase Eta ◽  
Pyrimidine Dimers ◽  
Dna Polymerase Eta ◽  
Y Family ◽  
Proliferating Cell

Human DNA polymeraseη(HsPolη) plays an important role in translesion synthesis (TLS), which allows for replication past DNA damage such as UV-inducedcis-syncyclobutane pyrimidine dimers (CPDs). Here, we characterized ApPolηfrom the thermophilic wormAlvinella pompejana, which inhabits deep-sea hydrothermal vent chimneys. ApPolηshares sequence homology with HsPolηand contains domains for binding ubiquitin and proliferating cell nuclear antigen. Sun-induced UV does not penetrateAlvinella'senvironment; however, this novel DNA polymerase catalyzed efficient and accurate TLS past CPD, as well as 7,8-dihydro-8-oxoguanine and isomers of thymine glycol induced by reactive oxygen species. In addition, we found that ApPolηis more thermostable than HsPolη, as expected from its habitat temperature. Moreover, the activity of this enzyme was retained in the presence of a higher concentration of organic solvents. Therefore, ApPolηprovides a robust, human-like Polηthat is more active after exposure to high temperatures and organic solvents.

DNA polymerase ι-dependent translesion replication of uracil containing cyclobutane pyrimidine dimers

DNA Repair ◽  
2006 ◽  
Vol 5 (2) ◽  
pp. 210-218 ◽  
Author(s):  
Alexandra Vaisman ◽  
Kohei Takasawa ◽  
Shigenori Iwai ◽  
Roger Woodgate
Keyword(s):  
Dna Polymerase ◽  
Cyclobutane Pyrimidine Dimers ◽  
Pyrimidine Dimers ◽  
Dna Polymerase Ι

scholarly journals p48 Activates a UV-Damaged-DNA Binding Factor and Is Defective in Xeroderma Pigmentosum Group E Cells That Lack Binding Activity

1998 ◽  
Vol 18 (7) ◽  
pp. 4391-4399 ◽  
Author(s):  
Byung Joon Hwang ◽  
Stephanie Toering ◽  
Uta Francke ◽  
Gilbert Chu
Keyword(s):  
Xeroderma Pigmentosum ◽  
Chromosome Region ◽  
Binding Activity ◽  
Single Amino Acid ◽  
Cyclobutane Pyrimidine Dimers ◽  
Pyrimidine Dimers ◽  
Binding Factor ◽  
Hit And Run ◽  
Damaged Dna

ABSTRACT A subset of xeroderma pigmentosum (XP) group E cells lack a factor that binds to DNA damaged by UV radiation. This factor can be purified to homogeneity as p125, a 125-kDa polypeptide. However, when cDNA encoding p125 is translated in vitro, only a small fraction binds to UV-damaged DNA, suggesting that a second factor is required for the activation of p125. We discovered that most hamster cell lines expressed inactive p125, which was activated in somatic cell hybrids containing human chromosome region 11p11.2-11cen. This region excludedp125 but included p48, which encodes a 48-kDa polypeptide known to copurify with p125 under some conditions. Expression of human p48 activated p125 binding in hamster cells and increased p125 binding in human cells. No such effects were observed from expression of p48 containing single amino acid substitutions from XP group E cells that lacked binding activity, demonstrating that the p48 gene is defective in those cells. Activation of p125 occurred by a “hit-and-run” mechanism, since the presence of p48 was not required for subsequent binding. Nevertheless, p48 was capable of forming a complex with p125 either bound to UV-damaged DNA or in free solution. It is notable that hamster cells fail to efficiently repair cyclobutane pyrimidine dimers in nontranscribed DNA and fail to express p48, which contains a WD motif with homology to proteins that reorganize chromatin. We propose that p48 plays a role in repairing lesions that would otherwise remain inaccessible in nontranscribed chromatin.

scholarly journals Yeast DNA Polymerase ζ Is an Efficient Extender of Primer Ends Opposite from 7,8-Dihydro-8-Oxoguanine and O6-Methylguanine

2003 ◽  
Vol 23 (4) ◽  
pp. 1453-1459 ◽  
Author(s):  
Lajos Haracska ◽  
Satya Prakash ◽  
Louise Prakash
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Polymerase ◽  
Uv Light ◽  
Dna Lesions ◽  
Geometric Distortion ◽  
Cyclobutane Pyrimidine Dimers ◽  
Genetic Studies ◽  
Abasic Sites ◽  
Dna Damaging Agents ◽  
Pyrimidine Dimers

ABSTRACT Genetic studies in Saccharomyces cerevisiae have indicated the requirement of DNA polymerase (Pol) ζ for mutagenesis induced by UV light and by other DNA damaging agents. However, on its own, Polζ is highly inefficient at replicating through DNA lesions; rather, it promotes their mutagenic bypass by extending from the nucleotide inserted opposite the lesion by another DNA polymerase. So far, such a role for Polζ has been established for cyclobutane pyrimidine dimers, (6-4) dipyrimidine photoproducts, and abasic sites. Here, we examine whether Polζ can replicate through the 7,8-dihydro-8-oxoguanine (8-oxoG) and O 6-methylguanine (m6G) lesions. We chose these two lesions for this study because the replicative polymerase, Polδ, can replicate through them, albeit weakly. We found that Polζ is very inefficient at inserting nucleotides opposite both these lesions, but it can efficiently extend from the nucleotides inserted opposite them by Polδ. Also, the most efficient bypass of 8-oxoG and m6G lesions occurs when Polδ is combined with Polζ, indicating a role for Polζ in extending from the nucleotides inserted opposite these lesions by Polδ. Thus, Polζ is a highly specialized polymerase that can proficiently extend from the primer ends opposite DNA lesions, irrespective of their degree of geometric distortion. Polζ, however, is unusually sensitive to geometric distortion of the templating residue, as it is highly inefficient at incorporating nucleotides even opposite the moderately distorting 8-oxoG and m6G lesions.

scholarly journals A Role for Histone H2B During Repair of UV-Induced DNA Damage in Saccharomyces cerevisiae

Genetics ◽  
2002 ◽  
Vol 160 (4) ◽  
pp. 1375-1387
Author(s):  
Emmanuelle M D Martini ◽  
Scott Keeney ◽  
Mary Ann Osley
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Chromatin Structure ◽  
Specific Effect ◽  
Cell Killing ◽  
Cyclobutane Pyrimidine Dimers ◽  
Histone H2b ◽  
Uv Sensitivity ◽  
Pyrimidine Dimers

Abstract To investigate the role of the nucleosome during repair of DNA damage in yeast, we screened for histone H2B mutants that were sensitive to UV irradiation. We have isolated a new mutant, htb1-3, that shows preferential sensitivity to UV-C. There is no detectable difference in bulk chromatin structure or in the number of UV-induced cis-syn cyclobutane pyrimidine dimers (CPD) between HTB1 and htb1-3 strains. These results suggest a specific effect of this histone H2B mutation in UV-induced DNA repair processes rather than a global effect on chromatin structure. We analyzed the UV sensitivity of double mutants that contained the htb1-3 mutation and mutations in genes from each of the three epistasis groups of RAD genes. The htb1-3 mutation enhanced UV-induced cell killing in rad1Δ and rad52Δ mutants but not in rad6Δ or rad18Δ mutants, which are defective in postreplicational DNA repair (PRR). When combined with other mutations that affect PRR, the histone mutation increased the UV sensitivity of strains with defects in either the error-prone (rev1Δ) or error-free (rad30Δ) branches of PRR, but did not enhance the UV sensitivity of a strain with a rad5Δ mutation. When combined with a ubc13Δ mutation, which is also epistatic with rad5Δ, the htb1-3 mutation enhanced UV-induced cell killing. These results suggest that histone H2B acts in a novel RAD5-dependent branch of PRR.

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