Environmental and Molecular Mutagenesis - Legacy Tobacco ...

12 1989 EMS Abstracts 26
Notes Temporal control of AP endonuclease !n human flbroblasts and Blooms syndrome cells . P .
Arenaz, S . Trevizo, P . Anaya, V . Pelaez and L . Winkfield, Dept . Biology, University of
Texas El Paso, El Paso, TX (USA)
Previous reports have indicated that DNA repair and replication are coordinately
regulated within the defined context of the cell cycle and that this temporal
regulation acts as a screen to ensure replicative fidelity . Certain putative DNA
repair deficient syndromes exhibit an aberrant pattern of gene expression of several
DNA repair enzymes . We have investigated the temporal regulation of AP endonuclease
in human fibroblasts and in Bloom's syndrome cells . Celie were synchronized by either
serum starvation or hydroxyurea and cells collected at various time points after
release from the block . DNA repair and replicative parameters were assayed using cell
extracts . AP endonuclease activity, measured in serum synchronized cells was maximal
at 18 h, 3 h prior to DNA synthesis and one hour prior to the fnduction of uracil DNA
g)ycosylase . This same pattern was observed for AP endonuclease activity after
hydroxyurea synchronization . In Bloom's syndrome cells, AP endonuclease was again
Induced prior to DNA synthesis . However, as reported before uracil DNA glycosylase
was induced concomitant with DNA synthesis . These data suggest that AP endonuclease
ia Induced within the defined context of the cell cycle in both normal human
fibroblasts and Bloom's cells . Furthermore, 1t appears that AP endonucleaoe and
uracil DNA glycosylase are not concomitantly controlled and suggests that there may be
different control signals involved in the induction of these enzymes .
Supported in part by NIH grant RR08012 and a grant from the University of Texas at E1
Paso .
http://legacy.library.ucsf.edu/tid/clb93d00/pdf
CHARACTERIZATION OF A DIRECT-ACTING MUTAGEN FORMED FROM N-NITROSOPIPERIDINE AND
PHOSPHATE WITH NEAR-ULTRAVIOLET LIGHT IRRADIATION
Sakae Arimoto, Hiromi Shimada, Satoko Ukawal, Masataka Mochizukil and Hikoya Hayatsu
Faculty of Pharmaceutical Sciences, Okayama University . Tsushima, Okayama 700, and
1Kyoritsu College of Pharmacy, Shibakoen, Minato-ku, Tokyo 105, Japan
Previously we found that direct-acting autagens can be formed from N-nitrosodialkylamines
on exposure to UVA . We have now isolated the product formed from N-nitrosopiperidine
(NPIP) and investigated its structure . NPIP (40 mM) in 20 mM sodium phosphate
at pH 7 .4 was irradiated with UVA (313-400 nm, 10 yN/mm2) for 3 hr . Direct-acting
mutagenicity of the solution on S . typhimurium TA 1535 was 44000 His+ revertants for
1 mmole equivalent of NPIP . The reaction mixture was freeze-dried and the residue was
extracted with methanol . The methanol extract was evaporated to dryness under reduced
pressure, and the residue was fractionated by HPLC on an ODS column . Direct-acting
mutagenicity was observed only in one UV-absorbing peak . The compound in this peak
fraction was identical to an authentic sample of a-phosphonooxy-NPIP in terms of the
retention time in HPLC, UV spectrum, sensitivity to phosphatase and the mutagenic
potency as determined on the basis of A231 units . Moreover, the 1H-NMR spectrum of the
isolated compound was identical to that of the authentic specimen . Thus, it waa
established that N-nitrosodialkylamines can be transformed into their 0-hydroxy phosphate
esters by the action of near UV light in the presence of inorganic phosphate .
This reaction represents a new, non-enzymatic activation of promutagenic N-nitrosodialkylamines
.
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ANALYSIS OF MUTATIONS INDUCED BY NEAR-UV RADIATION . J .D . Armstrong, M . Glattke, L .
Kohalmi and B .A . Kunz, Microbiology Department, The University of Manitoba, Winnipeg,
Manitoba, Canada R3T 2N2
Ultraviolet (UV) wavelengths found in solar radiation are autagenic and carcinogenic
. Attenuation by atmospheric ozone limits the total amount of solar UV in our
environment and restricts incident solar UV wavelengths to the naar-W (NUV : 300-400
nm) region so that virtually no UVC (200-280 nm) wavelengths are present . To begin
probing the mechanism(s) responsible for solar UV mutagenesis, we have used DNA sequencing
to characterize 120 autetlons induced in the SUP4-o gene of yeast by NUV
and have compared the resulting spectrum to that for 185 UVC-induced mutations . In
both cases, single base-pair substitutions accounted for approx . 90% of the induced
mutations but the fraction of double-pair changes was 3-fold greater for NW and all
double mutations were tandem events compared to only 330 for UVC . For each agent,
approx . 90% of the substitutions were transitions but NUV induced substantially more
G•C -> A•T events than UVC (88t vs . 681, respectively) . Of the substitutions that
could be assigned to the 5' or 3' base of particular dipyrimidines, 800 of those induced
by NUV occurred at the 3' base of S'-TC-3' or 5'-CC-3' sites compared to 60%
for UVC . In addition, S'-TT-3' sites that were hotapots for UVC were not targets for
NUV mutagenesis although hotspots at 5'-TC-3' sites coincided . Wavelengths involved
in photoreactivation of cyclobutane dimers or photolysis of [6-41 photoproducts, are
present in NUV radiation . On this basis, our data suggest that either (6-41 photophotoproducts,
or photoproducts resulting from photolysis of these lesions, are the
major premutational NW lesions . (Supported by NSERC Canada)
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