Initiation of Repair of DNA-Polypeptide Cross-Links by the UvrABC Nuclease
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- 作者：Irina G. Minko ; Andrew J. Kurtz ; Deborah L. Croteau ; Bennett Van Houten ; Thomas M. Harris ; and R. Stephen Lloyd
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：March 1, 2005
- 年：2005
- 卷：44
- 期：8
- 页码：3000 - 3009
- 全文大小：405K
- 年卷期：v.44,no.8(March 1, 2005)
- ISSN：1520-4995
文摘
Although the biochemical pathways that repair DNA-protein cross-links have not been clearlyelucidated, it has been proposed that the partial proteolysis of cross-linked proteins into smaller oligopeptidesconstitutes an initial step in removal of these lesions by nucleotide excision repair (NER). To test thevalidity of this repair model, several site-specific DNA-peptide and DNA-protein cross-links wereengineered via linkage at (1) an acrolein-derived 
-hydroxypropanodeoxyguanosine adduct and (2) anapurinic/apyrimidinic site, and the initiation of repair was examined in vitro using recombinant proteinsUvrA and UvrB from Bacillus caldotenax and UvrC from Thermotoga maritima. The polypeptides cross-linked to DNA were Lys-Trp-Lys-Lys, Lys-Phe-His-Glu-Lys-His-His-Ser-His-Arg-Gly-Tyr, and the 16kDa protein, T4 pyrimidine dimer glycosylase/apurinic/apyrimidinic site lyase. For the substrates examined,DNA incision required the coordinated action of all three proteins and occurred at the eighth phosphodiesterbond 5' to the lesion. The incision rates for DNA-peptide cross-links were comparable to or greater thanthat measured on fluorescein-adducted DNA, an excellent substrate for UvrABC. Incision rates weredependent on both the site of covalent attachment on the DNA and the size of the bound peptide.Importantly, incision of a DNA-protein cross-link occurred at a rate approximately 3.5-8-fold slowerthan the rates observed for DNA-peptide cross-links. Thus, direct evidence has been obtained indicatingthat (1) DNA-peptide cross-links can be efficiently incised by the NER proteins and (2) DNA-peptidecross-links are preferable substrates for this system relative to DNA-protein cross-links. These data suggestthat proteolytic degradation of DNA-protein cross-links may be an important processing step in facilitatingNER.
-hydroxypropanodeoxyguanosine adduct and (2) anapurinic/apyrimidinic site, and the initiation of repair was examined in vitro using recombinant proteinsUvrA and UvrB from Bacillus caldotenax and UvrC from Thermotoga maritima. The polypeptides cross-linked to DNA were Lys-Trp-Lys-Lys, Lys-Phe-His-Glu-Lys-His-His-Ser-His-Arg-Gly-Tyr, and the 16kDa protein, T4 pyrimidine dimer glycosylase/apurinic/apyrimidinic site lyase. For the substrates examined,DNA incision required the coordinated action of all three proteins and occurred at the eighth phosphodiesterbond 5' to the lesion. The incision rates for DNA-peptide cross-links were comparable to or greater thanthat measured on fluorescein-adducted DNA, an excellent substrate for UvrABC. Incision rates weredependent on both the site of covalent attachment on the DNA and the size of the bound peptide.Importantly, incision of a DNA-protein cross-link occurred at a rate approximately 3.5-8-fold slowerthan the rates observed for DNA-peptide cross-links. Thus, direct evidence has been obtained indicatingthat (1) DNA-peptide cross-links can be efficiently incised by the NER proteins and (2) DNA-peptidecross-links are preferable substrates for this system relative to DNA-protein cross-links. These data suggestthat proteolytic degradation of DNA-protein cross-links may be an important processing step in facilitatingNER.