Dependence of DNA鈥揚rotein Cross-Linking via Guanine Oxidation upon Local DNA Sequence As Studied by Restriction Endonuclease Inhibition
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- 作者：Amanda L. Madison ; Zitadel A. Perez ; Phuong To ; Tiffany Maisonet ; Eunice V. Rios ; Yuri Trejo ; Carmen Ochoa-Paniagua ; Anita Reno ; Eric D. A. Stemp
- 刊名：Biochemistry
- 出版年：2012
- 出版时间：January 10, 2012
- 年：2012
- 卷：51
- 期：1
- 页码：362-369
- 全文大小：335K
- 年卷期：v.51,no.1(January 10, 2012)
- ISSN：1520-4995
文摘
Oxidative damage plays a causative role in many diseases, and DNA鈥損rotein cross-linking is one important consequence of such damage. It is known that GG and GGG sites are particularly prone to one-electron oxidation, and here we examined how the local DNA sequence influences the formation of DNA鈥損rotein cross-links induced by guanine oxidation. Oxidative DNA鈥損rotein cross-linking was induced between DNA and histone protein via the flash quench technique, a photochemical method that selectively oxidizes the guanine base in double-stranded DNA. An assay based on restriction enzyme cleavage was developed to detect the cross-linking in plasmid DNA. Following oxidation of pBR322 DNA by flash quench, several restriction enzymes (PpuMI, BamHI, EcoRI) were then used to probe the plasmid surface for the expected damage at guanine sites. These three endonucleases were strongly inhibited by DNA鈥損rotein cross-linking, whereas the AT-recognizing enzyme AseI was unaffected in its cleavage. These experiments also reveal the susceptibility of different guanine sites toward oxidative cross-linking. The percent inhibition observed for the endonucleases, and their pBR322 cleavage sites, decreased in the order: PpuMI (5鈥?GGGTCCT-3鈥?and 5鈥?AGGACCC-3鈥? > BamHI (5鈥?GGATCC-3鈥? > EcoRI (5鈥?GAATTC-3鈥?, a trend consistent with the observed and predicted tendencies for guanine to undergo one-electron oxidation: 5鈥?GGG-3鈥?> 5鈥?GG-3鈥?> 5鈥?GA-3鈥? Thus, it appears that in mixed DNA sequences the guanine sites most vulnerable to oxidative cross-linking are those that are easiest to oxidize. These results further indicate that equilibration of the electron hole in the plasmid DNA occurs on a time scale faster than that of cross-linking.