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Title: DNA endonuclease activities on psoralen plus ultraviolet light treated DNA

Abstract

Activities of nuclear DNA endonucleases (Endos) from normal human lymphoblastoid cells on DNA treated with the DNA interstrand cross-linking agents 4,5'8-trimethyl psoralen (TMP) or 8-methoxypsoralen (MOP) plus long-wavelength (320-400 nm) ultraviolet light (UVA) were examined. Chromatin-associated DNA Endos were isolated from both cell lines and subjected to isoelectric focusing (IF). Each IF fraction was assayed for DNA Endo activity. Peaks of activity were pooled and assayed for activity on undamaged PM2 bacteriophage DNA and on PM2 DNA that had been treated with 15 ..mu..g/ml TMP or MOP in the dark and then exposed to UVA light. Unbound psoralen was removed by dialysis and a second dose of UVA light was given in order to increase the number of DNA cross-links. Two Endo activities were found which were active on TMP- and MOP-DNA: a major one, pI 4.6, which is also active on intercalated DNA, and a second, lesser one, pI 7.6, which is active on UVC (254 nm) light irradiated DNA. These results indicate that there are two different DNA Endos which act on both TMP- and MOP-treated DNA and that the major activity recognizes the intercalation of, and/or the cross-link produced by interaction of, psoralen with DNA.

Authors:
;
Publication Date:
Research Org.:
New Jersey Medical School, Newark
OSTI Identifier:
5416158
Report Number(s):
CONF-8604222-
Journal ID: CODEN: FEPRA; TRN: 86-030543
Resource Type:
Conference
Resource Relation:
Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States); Journal Volume: 45:3; Conference: 70. annual meeting of the Federation of American Society for Experimental Biology, St. Louis, MO, USA, 13 Apr 1986
Country of Publication:
United States
Language:
English
Subject:
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.; DNA; GENETIC RADIATION EFFECTS; ENDONUCLEASES; ENZYME ACTIVITY; PSORALEN; GENETIC EFFECTS; BACTERIOPHAGES; CROSS-LINKING; ULTRAVIOLET RADIATION; ANTICOAGULANTS; BIOLOGICAL EFFECTS; BIOLOGICAL RADIATION EFFECTS; CHEMICAL REACTIONS; COUMARINS; DNA-ASE; DRUGS; ELECTROMAGNETIC RADIATION; ENZYMES; ESTERASES; HEMATOLOGIC AGENTS; HETEROCYCLIC COMPOUNDS; HYDROLASES; MICROORGANISMS; NUCLEIC ACIDS; ORGANIC COMPOUNDS; ORGANIC OXYGEN COMPOUNDS; PARASITES; PHOSPHODIESTERASES; POLYMERIZATION; RADIATION EFFECTS; RADIATIONS; VIRUSES; 560111* - Radiation Effects on Biochemicals- In Vitro- (-1987); 560301 - Chemicals Metabolism & Toxicology- Cells- (-1987)

Citation Formats

Lambert, M.W., and Clark, M. DNA endonuclease activities on psoralen plus ultraviolet light treated DNA. United States: N. p., 1986. Web.
Lambert, M.W., & Clark, M. DNA endonuclease activities on psoralen plus ultraviolet light treated DNA. United States.
Lambert, M.W., and Clark, M. 1986. "DNA endonuclease activities on psoralen plus ultraviolet light treated DNA". United States. doi:.
@article{osti_5416158,
title = {DNA endonuclease activities on psoralen plus ultraviolet light treated DNA},
author = {Lambert, M.W. and Clark, M.},
abstractNote = {Activities of nuclear DNA endonucleases (Endos) from normal human lymphoblastoid cells on DNA treated with the DNA interstrand cross-linking agents 4,5'8-trimethyl psoralen (TMP) or 8-methoxypsoralen (MOP) plus long-wavelength (320-400 nm) ultraviolet light (UVA) were examined. Chromatin-associated DNA Endos were isolated from both cell lines and subjected to isoelectric focusing (IF). Each IF fraction was assayed for DNA Endo activity. Peaks of activity were pooled and assayed for activity on undamaged PM2 bacteriophage DNA and on PM2 DNA that had been treated with 15 ..mu..g/ml TMP or MOP in the dark and then exposed to UVA light. Unbound psoralen was removed by dialysis and a second dose of UVA light was given in order to increase the number of DNA cross-links. Two Endo activities were found which were active on TMP- and MOP-DNA: a major one, pI 4.6, which is also active on intercalated DNA, and a second, lesser one, pI 7.6, which is active on UVC (254 nm) light irradiated DNA. These results indicate that there are two different DNA Endos which act on both TMP- and MOP-treated DNA and that the major activity recognizes the intercalation of, and/or the cross-link produced by interaction of, psoralen with DNA.},
doi = {},
journal = {Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)},
number = ,
volume = 45:3,
place = {United States},
year = 1986,
month = 3
}

Conference:
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  • A new method is proposed to inhibit semiconservative DNA synthesis in cultured cells while DNA repair synthesis is being measured. The cells are treated with the DNA-crosslinking agent Trioxalen (4,5,8-trimethylpsoralen) plus near-ultraviolet light, and consequently 99.5% inhibition of replicative DNA synthesis is achieved. Additional DNA-damaging agents induce thymidine incorporation into the double-stranded regions of the DNA. The new method gave results very similar to those obtained with the benzoylated naphthoylated DEAE (BND) cellulose method using three human fibroblast strains, of which one had deficient capacity for DNA repair synthesis following treatment with gamma rays and methyl methanesulfonate. The advantages ofmore » the new method are simplicity and rapidity, as well as the high extent to which replicative DNA synthesis is inhibited.« less
  • A new method is proposed to inhibit semiconservative DNA synthesis in cultured cells while DNA repair synthesis is being measured. The cells are treated with the DNA-crosslinking agent Trioxalen (4,5,8-trimethylpsoralen) plus near-ultraviolet light, and consequently 99.5% inhibition of replicative DNA synthesis is achieved. Additional DNA-damaging agents induce thymidine incorporation into the double-stranded regions of the DNA. The new method gave results very similar to those obtained with the benzoylated naphthoylated DEAE (BND) cellulose method using three human fibroblast strains, of which one had deficient capacity for DNA repair synthesis following treatment with ..gamma.. rays and methyl methanesulfonate. The advantages ofmore » the new method are simplicity and rapidity, as well as the high extent to which replicative DNA synthesis is inhibited.« less
  • The relative contributions of excision and recombination in the repair of damage by 8-methoxypsoralen (8-MOP) plus black light to Bacillus subtilis were studied. The results indicate that the pyrimidine dimer excision system and a recombination pathway are probably both involved in repair of lethal damage to cells exposed in vivo to 8-MOP plus black light, but repair is not very efficient. Transforming DNA exposed in vitro to 8-MOP plus black light was inactivated mainly by crosslinks rather than by monoadducts, and was repaired predominantly by an incision-dependent process. There was very little demonstrable damage-induced recombination in transforming DNA.
  • An endonuclease that makes single polynucleotide chain scissions in uv- irradiated DNA has been purified from Escherichia coli. The activity has the following properties: (1) unirradiated DNA is attacked very little if at all; single-stranded DNA is not attacked, whether irradiated or not; there is no requirement for divalent cations, and the activity is not affected by the addition of EDTA; the pH optimum is approximately 7; the activity is inhibited by 1 M NaCl, single-stranded DNA, transfer RNA, and unirradiated double-stranded DNA; the sedimentation coefficient, S$sub 20$,/sub W'/ is approximately 2.6; it is a basic protein. The enzyme ismore » tentatively named E. coli endonuclease III. The physiological function of the endonuclease has not yet been established. (auth)« less