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(Received for publication, April 1, 1996, and in revised form, July 17, 1996)
From the Institut de Pharmacologie et de Biologie Structurale,
CNRS, UPR 9062, 205 route de Narbonne, 31077 Toulouse, France
Nucleotide excision repair (NER) was measured in
human cell extracts incubated with either supercoiled or linearized
damaged plasmid DNA as repair substrate. NER, as quantified by the
extent of repair synthesis activity, was reduced by up to 80% in the
case of linearized plasmid DNA when compared with supercoiled DNA. An
excess of undamaged linearized plasmid in the repair mixture did not
interfere with DNA repair synthesis activity on a supercoiled damaged
plasmid, indicating a cis-acting inhibiting effect.
In contrast, gaps on circular or linearized plasmids were filled in
identically by the DNA polymerases operating in the extracts. When the
extent of damage-dependent incision activity was measured,
a ~70% reduction of repair incision activity by human cell extract
was observed on linearized damaged plasmids. Recessed, protruding, or
blunt ends were similarly inhibitory.
NER activity was partly restored when the extracts were preincubated
with autoimmune human sera containing antibodies against the nuclear
DNA end-binding heterodimer Ku. In addition, the inhibition of repair
activity on linear damaged plasmids was released in extracts from
rodent cells deficient in Ku activity but not in extracts from murine
scid cells devoid of Ku-associated
DNA-dependent kinase activity.
Volume 271, Number 44,
Issue of November 1, 1996
pp. 27601-27607
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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