Linked Life Data

7093202

Source:http://linkedlifedata.com/resource/pubmed/id/7093202

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
10
pubmed:dateCreated
1982-9-10
pubmed:abstractText
We have extended our permeable cell system for measuring DNA excision repair [Roberts, J. D., & Lieberman, M. W. (1979) Biochemistry 18, 4499-4505] so that steps of the repair process, beginning with incision and extending at least through the "rearrangement" of repaired nucleosomes which follows repair synthesis, all take place in permeable cells. In the revised protocol, human fibroblasts are made permeable, damaged with UV or chemicals in suspension, and incubated with a reaction mix containing ATP and the four deoxyribonucleoside triphosphates, one of which is labeled with 32P. By reducing the exogenous dNTP concentration to 3 microM and including 15 mM KCl in the reaction mixture, we have greatly reduced background incorporation in undamaged cells without significantly reducing repair synthesis. This permits us to measure repair synthesis without separating it from replicative synthesis by isopycnic centrifugation. Repair synthesis in this system is very similar to that occurring in intact cells: in response to DNA damage, nucleotides are incorporated into DNA of parental density (when analyzed by the BrdUrd density shift technique), incorporation increases with increasing DNA damage, synthesis is dependent on the presence of all four dNTPs, and the system accurately reflects the genetic UV repair deficiency of xeroderma pigmentosum (XP) cells. Furthermore, as has been observed in intact cells, repair-incorporated nucleotides in these permeable cells are initially overrepresented in staphylococcal nuclease sensitive regions of chromatin and are subsequently redistributed to give a nearly uniform distribution between nuclease-sensitive and -resistant regions. The UV dose curve of permeable cells differs somewhat from that of intact cells; however, the dose differs somewhat from that of intact cells; however, the dose curve for permeable cells treated with N-methyl-N-nitrosourea is very similar to that of intact cells. Repair synthesis in UV-damaged, permeable normal and XP cells is stimulated by addition of Micrococcus luteus UV endonuclease, indicating that the damaged DNA is accessible to exogenous repair enzymes and suggesting that incision, or an obligatory preincision step, is rate limiting for excision repair in these permeable cells. Repair synthesis in this system is inhibited by aphidicolin, but not by high levels of dideoxy-TTP, suggesting involvement of DNA polymerase alpha in excision repair. Novobiocin is also inhibitory alpha and the HeLa cell type II DNA topoisomerase.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
May
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
11
pubmed:volume
21
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
2557-64
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
1982
pubmed:articleTitle
Characterization of deoxyribonucleic acid repair synthesis in permeable human fibroblasts.
pubmed:publicationType
Journal Article, Comparative Study, Research Support, U.S. Gov't, P.H.S., Research Support, Non-U.S. Gov't