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. 1993 Aug 1;293(Pt 3):825–828. doi: 10.1042/bj2930825

Kinetics and mechanism of DNA repair: an automated programmable apparatus for fast time-resolved studies of the repair of mammalian DNA after u.v. irradiation.

C W Wharton 1, R A Meldrum 1, C Reason 1, J Boone 1, W Lester 1
PMCID: PMC1134442  PMID: 8352751

Abstract

An automated apparatus, designed and constructed for use in fast time-resolved studies of mammalian DNA repair after u.v. irradiation, is described. The methodology makes use of caged DNA-break-trapping reagents, e.g. [alpha-32P]dideoxyadenosine triphosphate [see Meldrum, Shall, Trentham and Wharton (1990) Biochem J. 266, 885-890] and the apparatus incorporates excimer lasers both for the delivery of u.v. damage and for the photoactivation of the caged reagents. The design is based on a sample-changing turntable which permits the sequential irradiation and quenching of samples. A maximum of eight samples can be processed in a single experiment, the sequence of events being programmed on a microcomputer, which permits a very generalized experimental sequence. Pipettes for addition of cells, nucleotides and quenching agent are driven pneumatically. A pair of pneumatically operated platinum electrodes allows electroporation of cells for loading of negatively charged reagents prior to irradiation. The time resolution of the apparatus is dependent upon the experimental scheme used and can be very short (e.g. 1 ms) when caged reagents are used; a more usual period is 1 s for the shortest incubation.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Chu G., Hayakawa H., Berg P. Electroporation for the efficient transfection of mammalian cells with DNA. Nucleic Acids Res. 1987 Feb 11;15(3):1311–1326. doi: 10.1093/nar/15.3.1311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Collins A. R., Downes C. S., Johnson R. T. Introduction: an integrated view of inhibited repair. Nucleic Acids Symp Ser. 1984;(13):1–11. [PubMed] [Google Scholar]
  3. Dresler S. L., Kimbro K. S. 2',3'-Dideoxythymidine 5'-triphosphate inhibition of DNA replication and ultraviolet-induced DNA repair synthesis in human cells: evidence for involvement of DNA polymerase delta. Biochemistry. 1987 May 19;26(10):2664–2668. doi: 10.1021/bi00384a002. [DOI] [PubMed] [Google Scholar]
  4. Erixon K., Ahnström G. Single-strand breaks in DNA during repair of UV-induced damage in normal human and xeroderma pigmentosum cells as determined by alkaline DNA unwinding and hydroxylapatite chromatography: effects of hydroxyurea, 5-fluorodeoxyuridine and 1-beta-D-arabinofuranosylcytosine on the kinetics of repair. Mutat Res. 1979 Feb;59(2):257–271. doi: 10.1016/0027-5107(79)90164-7. [DOI] [PubMed] [Google Scholar]
  5. Madhani H. D., Bohr V. A., Hanawalt P. C. Differential DNA repair in transcriptionally active and inactive proto-oncogenes: c-abl and c-mos. Cell. 1986 May 9;45(3):417–423. doi: 10.1016/0092-8674(86)90327-2. [DOI] [PubMed] [Google Scholar]
  6. Meldrum R. A., Shall S., Trentham D. R., Wharton C. W. Kinetics and mechanism of DNA repair. Preparation, purification and some properties of caged dideoxynucleoside triphosphates. Biochem J. 1990 Mar 15;266(3):885–890. [PMC free article] [PubMed] [Google Scholar]
  7. Mellon I., Hanawalt P. C. Induction of the Escherichia coli lactose operon selectively increases repair of its transcribed DNA strand. Nature. 1989 Nov 2;342(6245):95–98. doi: 10.1038/342095a0. [DOI] [PubMed] [Google Scholar]
  8. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Schulte-Frohlinde D., Simic M. G., Görner H. Laser-induced strand break formation in DNA and polynucleotides. Photochem Photobiol. 1990 Dec;52(6):1137–1151. doi: 10.1111/j.1751-1097.1990.tb08453.x. [DOI] [PubMed] [Google Scholar]
  10. Squires S., Johnson R. T., Collins A. R. Initial rates of DNA incision in UV-irradiated human cells: differences between normal, xeroderma pigmentosum and tumour cells. Mutat Res. 1982 Aug;95(2-3):389–404. doi: 10.1016/0027-5107(82)90273-1. [DOI] [PubMed] [Google Scholar]
  11. Walker J. W., Reid G. P., Trentham D. R. Synthesis and properties of caged nucleotides. Methods Enzymol. 1989;172:288–301. doi: 10.1016/s0076-6879(89)72019-x. [DOI] [PubMed] [Google Scholar]

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