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Title: A combinatorial system to examine the enzymatic repair of multiply damaged DNA substrates
Abstract DNA damage drives genetic mutations that underlie the development of cancer in humans. Multiple pathways have been described in mammalian cells which can repair this damage. However, most work to date has focused upon single lesions in DNA. We present here a combinatorial system which allows assembly of duplexes containing single or multiple types of damage by ligating together six oligonucleotides containing damaged or modified bases. The combinatorial system has dual fluorescent labels allowing examination of both strands simultaneously, in order to study interactions or competition between different DNA repair pathways. Using this system, we demonstrate how repair of oxidative damage in one DNA strand can convert a mispaired T:G deamination intermediate into a T:A mutation. We also demonstrate that slow repair of a T:G mispair, relative to a U:G mispair, by the human methyl-binding domain 4 DNA glycosylase provides a competitive advantage to competing repair pathways, and could explain why CpG dinucleotides are hotspots for C to T mutations in human tumors. Data is also presented that suggests repair of closely spaced lesions in opposing strands can be repaired by a combination of short and long-patch base excision repair and simultaneous repair of multiply damage sites can potentially lead to lethal double strand breaks.  more » « less
Award ID(s):
2129617
NSF-PAR ID:
10388079
Author(s) / Creator(s):
; ; ; ; ; ; ;
Date Published:
Journal Name:
Nucleic Acids Research
Volume:
50
Issue:
13
ISSN:
0305-1048
Page Range / eLocation ID:
7406 to 7419
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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