Linked Life Data

16274707

Source:http://linkedlifedata.com/resource/pubmed/id/16274707

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1-2
pubmed:dateCreated
2006-2-13
pubmed:abstractText
Chronic inflammation is associated with a variety of human diseases, including cancer, with one possible mechanistic link involving over-production of nitric oxide (NO*) by activated macrophages. Subsequent reaction of NO* with superoxide in the presence of carbon dioxide yields nitrosoperoxycarbonate (ONOOCO2-), a strong oxidant that reacts with guanine in DNA to form a variety of oxidation and nitration products, such 2'-deoxy-8-oxoguanosine. Alternatively, the reaction of NO and O2 leads to the formation of N2O3, a nitrosating agent that causes nucleobase deamination to form 2'-deoxyxanthosine (dX) and 2'-deoxyoxanosine (dO) from dG; 2'-deoxyinosine (dI) from dA; and 2'-deoxyuridine (dU) from dC, in addition to abasic sites and dG-dG cross-links. The presence of both ONOOCO2- and N2O3 at sites of inflammation necessitates definition of the relative roles of oxidative and nitrosative DNA damage in the genetic toxicology of inflammation. To this end, we sought to develop enzymatic probes for oxidative and nitrosative DNA lesions as a means to quantify the two types of DNA damage in in vitro DNA damage assays, such as the comet assay and as a means to differentially map the lesions in genomic DNA by the technique of ligation-mediated PCR. On the basis of fragmentary reports in the literature, we first systematically assessed the recognition of dX and dI by a battery of DNA repair enzymes. Members of the alkylpurine DNA glycosylase family (E. coli AlkA, murine Aag, and human MPG) all showed repair activity with dX (k(cat)/Km 29 x 10(-6), 21 x 10(-6), and 7.8 x 10(-6) nM(-1) min(-1), respectively), though the activity was considerably lower than that of EndoV (8 x 10(-3) nM(-1) min(-1)). Based on these results and other published studies, we focused the development of enzymatic probes on two groups of enzymes, one with activity against oxidative damage (formamidopyrimidine-DNA glycosylase (Fpg); endonuclease III (EndoIII)) and the other with activity against nucleobase deamination products (uracil DNA glycosylase (Udg); AlkA). These combinations were assessed for recognition of DNA damage caused by N2O3 (generated with a NO*/O2 delivery system) or ONOOCO2- using a plasmid nicking assay and by LC-MS analysis. Collectively, the results indicate that a combination of AlkA and Udg react selectively with DNA containing only nitrosative damage, while Fpg and EndoIII react selectively with DNA containing oxidative base lesions caused by ONOOCO2-. The results suggest that these enzyme combinations can be used as probes to define the location and quantity of the oxidative and nitrosative DNA lesions produced by chemical mediators of inflammation in systems, such as the comet assay, ligation-mediated polymerase chain reaction, and other assays of DNA damage and repair.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Feb
pubmed:issn
0027-5107
pubmed:author
pubmed:issnType
Print
pubmed:day
22
pubmed:volume
594
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
120-34
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
2006
pubmed:articleTitle
Development of enzymatic probes of oxidative and nitrosative DNA damage caused by reactive nitrogen species.
pubmed:affiliation
Biological Engineering Division NE47-277, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
pubmed:publicationType
Journal Article, Research Support, N.I.H., Extramural