Abstract:
:Quantitating relative (32)P-band intensity in gels is desired, e.g., to study primer-extension kinetics of DNA polymerases (DNAPs). Following imaging, multiple (32)P-bands are often present in lanes. Though individual bands appear by eye to be simple and well-resolved, scanning reveals they are actually skewed-Gaussian in shape and neighboring bands are overlapping, which complicates quantitation, because slower migrating bands often have considerable contributions from the trailing edges of faster migrating bands. A method is described to accurately quantitate adjacent (32)P-bands, which relies on having a standard: a simple skewed-Gaussian curve from an analogous pure, single-component band (e.g., primer alone). This single-component scan/curve is superimposed on its corresponding band in an experimentally determined scan/curve containing multiple bands (e.g., generated in a primer-extension reaction); intensity exceeding the single-component scan/curve is attributed to other components (e.g., insertion products). Relative areas/intensities are determined via pixel analysis, from which relative molarity of components is computed. Common software is used. Commonly used alternative methods (e.g., drawing boxes around bands) are shown to be less accurate. Our method was used to study kinetics of dNTP primer-extension opposite a benzo[a]pyrene-N(2)-dG-adduct with four DNAPs, including Sulfolobus solfataricus Dpo4 and Sulfolobus acidocaldarius Dbh. Vmax/Km is similar for correct dCTP insertion with Dpo4 and Dbh. Compared to Dpo4, Dbh misinsertion is slower for dATP (∼20-fold), dGTP (∼110-fold) and dTTP (∼6-fold), due to decreases in Vmax. These findings provide support that Dbh is in the same Y-Family DNAP class as eukaryotic DNAP κ and bacterial DNAP IV, which accurately bypass N(2)-dG adducts, as well as establish the scan-method described herein as an accurate method to quantitate relative intensity of overlapping bands in a single lane, whether generated from (32)P-signals or by other means (e.g., staining).
journal_name
DNA Repair (Amst)journal_title
DNA repairauthors
Sholder G,Loechler ELdoi
10.1016/j.dnarep.2014.10.001subject
Has Abstractpub_date
2015-01-01 00:00:00pages
97-103eissn
1568-7864issn
1568-7856pii
S1568-7864(14)00245-6journal_volume
25pub_type
杂志文章相关文献
DNA REPAIR文献大全abstract::Comparison of the clinical and cellular phenotypes of different genomic instability syndromes provides new insights into functional links in the complex network of the DNA damage response. A prominent example of this principle is provided by examination of three such disorders: ataxia-telangiectasia (A-T) caused by la...
journal_title:DNA repair
pub_type: 杂志文章,评审
doi:10.1016/j.dnarep.2004.04.009
更新日期:2004-08-01 00:00:00
abstract::Several types of DNA lesion are induced after ionizing irradiation (IR) of which double strand breaks (DSBs) are expected to be the most lethal, although single strand breaks (SSBs) and DNA base damages are quantitatively in the majority. Proteins of the base excision repair (BER) pathway repair these numerous lesions...
journal_title:DNA repair
pub_type: 杂志文章
doi:10.1016/j.dnarep.2008.11.008
更新日期:2009-03-01 00:00:00
abstract::Cells carrying deletions of genes encoding H-class ribonucleases display elevated rates of chromosome instability. The role of these enzymes is to remove RNA-DNA associations including persistent mRNA-DNA hybrids (R-loops) formed during transcription, and ribonucleotides incorporated into DNA during replication. RNase...
journal_title:DNA repair
pub_type: 杂志文章
doi:10.1016/j.dnarep.2017.02.012
更新日期:2017-04-01 00:00:00
abstract::Detection of γ-H2AX foci as a measure of DNA double strand break induction and repair provides the basis of a rapid approach to establish individual radiosensitivity. However, the assignment of criteria to define increased radiosensitivity is not straightforward. Experimental end points, analytical methods and prolife...
journal_title:DNA repair
pub_type: 杂志文章
doi:10.1016/j.dnarep.2013.07.002
更新日期:2013-10-01 00:00:00
abstract::Mice defective for the Polk gene, which encodes DNA polymerase kappa, are viable and do not manifest obvious phenotypes. The present studies document a spontaneous mutator phenotype in Polk(-/-) mice. The initial indication of enhanced spontaneous mutations in these mice came from the serendipitous observation of a po...
journal_title:DNA repair
pub_type: 杂志文章
doi:10.1016/j.dnarep.2009.09.003
更新日期:2009-12-03 00:00:00
abstract::AA8 Chinese hamster ovary cells were treated with halogenated nucleosides analogues of thymidine, namely CldU, 5-iodo-2'-deoxyuridine (IdU), and 5-bromo-2'-deoxyuridine (BrdU), following different experimental protocols. The purpose was to see whether incorporation of exogenous pyrimidine analogues into DNA could inte...
journal_title:DNA repair
pub_type: 杂志文章
doi:10.1016/s1568-7864(03)00044-2
更新日期:2003-06-11 00:00:00
abstract::Thymidylate deprivation brings about "thymineless death" in prokaryotes and eukaryotes. Although the precise mechanism for thymineless death has remained elusive, inhibition of the enzyme thymidylate synthase (TS), which catalyzes the de novo synthesis of TMP, has served for many years as a basis for chemotherapeutic ...
journal_title:DNA repair
pub_type: 杂志文章
doi:10.1016/j.dnarep.2008.06.006
更新日期:2008-10-01 00:00:00
abstract::Proteins that act on DNA, or are in close proximity to it, can become inadvertently crosslinked to DNA and form highly toxic lesions, known as DNA-protein crosslinks (DPCs). DPCs are generated by different chemotherapeutics, environmental or endogenous sources of crosslinking agents, or by lesions on DNA that stall th...
journal_title:DNA repair
pub_type: 杂志文章
doi:10.1016/j.dnarep.2020.102924
更新日期:2020-10-01 00:00:00
abstract::DNA repair is often a complex, multi-component, multi-step process; this makes detailed kinetic analysis of the different steps of repair a challenging task using standard biochemical methods. At the same time, single-molecule methods are well-suited for extracting kinetic information despite time-averaging due to dif...
journal_title:DNA repair
pub_type: 杂志文章,评审
doi:10.1016/j.dnarep.2014.02.018
更新日期:2014-08-01 00:00:00
abstract::The DNA polymerase beta (Pol beta) null background renders mouse embryonic fibroblast (MEF) cells base excision repair deficient and hyper-mutagenic upon treatment with the monofunctional alkylating agent, methyl methanesulfonate (MMS). This effect involves an increase in all types of base substitutions, with a modest...
journal_title:DNA repair
pub_type: 杂志文章
doi:10.1016/j.dnarep.2005.05.002
更新日期:2005-09-28 00:00:00
abstract::Base excision repair (BER) of damaged or inappropriate bases in DNA has been reported to take place by single nucleotide insertion or through incorporation of several nucleotides, termed short-patch and long-patch repair, respectively. We found that extracts from proliferating and non-proliferating cells both had capa...
journal_title:DNA repair
pub_type: 杂志文章
doi:10.1016/j.dnarep.2009.04.002
更新日期:2009-07-04 00:00:00
abstract::Huntington disease (HD) is associated with an unstable trinucleotide CAG.CTG repeat expansion. Although the repeat length is inversely correlated with the age-at-onset of symptoms, variability between patients who have inherited the same HD repeat length clearly suggests that other factors influence this aspect of the...
journal_title:DNA repair
pub_type: 杂志文章
doi:10.1016/j.dnarep.2007.01.002
更新日期:2007-06-01 00:00:00
abstract::Ataxia Telangiectasia (A-T) is a progressive childhood disorder characterized most notably by cerebellar degeneration and predisposition to cancer. A-T is caused by mutations in the kinase ATM, a master regulator of the DNA double-strand break response. In addition to DNA-damage signaling defects, A-T cells display mi...
journal_title:DNA repair
pub_type: 杂志文章
doi:10.1016/j.dnarep.2013.11.002
更新日期:2014-01-01 00:00:00
abstract::Plant mitochondrial and chloroplast genomes encode essential proteins for oxidative phosphorylation and photosynthesis. For proper cellular function, plant organelles must ensure genome integrity. Although plant organelles repair damaged DNA using the multi-enzyme Base Excision Repair (BER) pathway, the details of thi...
journal_title:DNA repair
pub_type: 杂志文章
doi:10.1016/j.dnarep.2018.02.010
更新日期:2018-05-01 00:00:00
abstract::Gene amplification, a key mechanism for oncogene activation and drug resistance in tumour cells, involves the generation and joining of DNA double-strand breaks. Amplified DNA can be carried either on intra-chromosomal arrays or on extra-chromosomal elements (double minutes). We previously showed that, in rodent cells...
journal_title:DNA repair
pub_type: 杂志文章
doi:10.1016/j.dnarep.2008.08.015
更新日期:2009-01-01 00:00:00
abstract::Nucleotide excision repair (NER), cell cycle regulation and apoptosis are major defence mechanisms against the carcinogenic effects of UVB radiation. NER eliminates UVB-induced DNA photolesions via two subpathways: global genome repair (GGR) and transcription-coupled repair (TCR). In a previous study, we found UVB-ind...
journal_title:DNA repair
pub_type: 杂志文章
doi:10.1016/j.dnarep.2004.08.008
更新日期:2005-01-02 00:00:00
abstract::RAre DAmage and Repair sequencing (RADAR-seq) is a highly adaptable sequencing method that enables the identification and detection of rare DNA damage events for a wide variety of DNA lesions at single-molecule resolution on a genome-wide scale. In RADAR-seq, DNA lesions are replaced with a patch of modified bases tha...
journal_title:DNA repair
pub_type: 杂志文章
doi:10.1016/j.dnarep.2019.06.007
更新日期:2019-08-01 00:00:00
abstract::O(6)-Alkylguanine-DNA alkyltransferase (MGMT) repairs DNA adducts that result from alkylation at the O(6) position of guanine. These lesions are mutagenic and toxic and can be produced by a variety of agents including the tobacco-specific nitrosamines, carcinogens present in cigarette smoke. Here, we review some of ou...
journal_title:DNA repair
pub_type: 杂志文章,评审
doi:10.1016/j.dnarep.2007.03.022
更新日期:2007-08-01 00:00:00
abstract::This article is an extension of previously published papers that periodically update a database of mouse mutant strains that are phenotypically defective in biological responses to DNA damage. Most of the mice listed in the database were generated by conventional targeted gene replacement technologies. ...
journal_title:DNA repair
pub_type: 杂志文章
doi:10.1016/s1568-7864(03)00005-3
更新日期:2003-05-13 00:00:00
abstract::To investigate involvement of DNA mismatch repair in the response to short-wave ultraviolet (UVC) light, we compared UVC-induced mutant frequencies and mutational spectra at the Hprt gene between wild type and mismatch-repair-deficient mouse embryonic stem (ES) cells. Whereas mismatch repair gene status did not signif...
journal_title:DNA repair
pub_type: 杂志文章
doi:10.1016/j.dnarep.2006.06.005
更新日期:2006-11-08 00:00:00
abstract::Microscopically visible gammaH2AX foci signify the presence of DNA double-strand breaks (dsbs) in irradiated cells. However, large foci are also observed in untreated tumour cells, and high numbers reduce the sensitivity for detecting drug or radiation-induced DNA breaks. SW756 cervical carcinoma cells that express ab...
journal_title:DNA repair
pub_type: 杂志文章
doi:10.1016/j.dnarep.2006.05.040
更新日期:2006-08-13 00:00:00
abstract::Artemis and PALF (also called APLF) appear to be among the primary nucleases involved in non-homologous end joining (NHEJ) and responsible for most nucleolytic end processing in NHEJ. About 60% of NHEJ events show an alignment of the DNA ends that use 1 or 2bp of microhomology (MH) between the two DNA termini. Thus, M...
journal_title:DNA repair
pub_type: 杂志文章,评审
doi:10.1016/j.dnarep.2014.02.006
更新日期:2014-05-01 00:00:00
abstract::We had shown previously that DNA polymerase beta (beta-pol) null mouse fibroblasts, deficient in base excision repair (BER), are hypersensitive to monofunctional methylating agents but not to hydrogen peroxide (H2O2). This is surprising because beta-pol is thought to be involved in BER of oxidative as well as methylat...
journal_title:DNA repair
pub_type: 杂志文章
doi:10.1016/s1568-7864(02)00008-3
更新日期:2002-04-29 00:00:00
abstract::There is an increasing demand for phenotyping assays in the field of human functional genetics. DNA repair activity is representative of this functional approach, being seen as a valuable biomarker related to cancer risk. Repair activity is evaluated by incubating a cell extract with a DNA substrate containing lesions...
journal_title:DNA repair
pub_type: 杂志文章
doi:10.1016/j.dnarep.2013.07.011
更新日期:2013-11-01 00:00:00
abstract::Multiple DNA polymerases participate in somatic hypermutation of immunoglobulin (Ig) genes. Mutations at A/T are largely dependent on DNA polymerase eta (POLH) whereas mutations at C/G appear to be generated by several DNA polymerases. We have previously shown that mice expressing a catalytically inactive POLQ (Polq-i...
journal_title:DNA repair
pub_type: 杂志文章
doi:10.1016/j.dnarep.2006.06.006
更新日期:2006-11-08 00:00:00
abstract::Structure-prone DNA repeats are common components of genomic DNA in all kingdoms of life. In humans, these repeats are linked to genomic instabilities that result in various hereditary disorders, including many cancers. It has long been known that DNA repeats are not only highly polymorphic in length but can also caus...
journal_title:DNA repair
pub_type: 杂志文章,评审
doi:10.1016/j.dnarep.2015.04.020
更新日期:2015-08-01 00:00:00
abstract::Telomeres play an important role in protecting the ends of chromosomes and preventing chromosome fusion. We have previously demonstrated that double-strand breaks near telomeres in mammalian cells result in either the addition of a new telomere at the site of the break, termed chromosome healing, or sister chromatid f...
journal_title:DNA repair
pub_type: 杂志文章
doi:10.1016/j.dnarep.2008.04.004
更新日期:2008-08-02 00:00:00
abstract::Exposure to ionizing radiation results in a variety of genome rearrangements that have been linked to tumor formation. Many of these rearrangements are thought to arise from the repair of double-strand breaks (DSBs) by several mechanisms, including homologous recombination (HR) between repetitive sequences dispersed t...
journal_title:DNA repair
pub_type: 杂志文章
doi:10.1016/j.dnarep.2008.02.003
更新日期:2008-05-03 00:00:00
abstract::Eukaryotic cells repair ultraviolet light (UV)- and chemical carcinogen-induced DNA strand-distorting damage through the nucleotide excision repair (NER) pathway. Concurrent activation of the DNA damage checkpoints is also required to arrest the cell cycle and allow time for NER action. Recent studies uncovered critic...
journal_title:DNA repair
pub_type: 杂志文章,评审
doi:10.1016/j.dnarep.2009.01.011
更新日期:2009-04-05 00:00:00
abstract::Methylation of the O(6)-methylguanine-DNA-methyltransferase (MGMT) promoter is associated with G:C to A:T transitions in the p53 gene in various human cancers, including lung cancer. In tumors with p53 mutation, MGMT promoter methylation is more common in advanced tumors than in early tumors. However, in tumors with w...
journal_title:DNA repair
pub_type: 杂志文章
doi:10.1016/j.dnarep.2008.04.016
更新日期:2008-08-02 00:00:00