Unusual Structural Features of Hydantoin Lesions Translate into Efficient Recognition by Escherichia coli Fpg

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• 作者：Nirmala Krishnamurthy ; James G. Muller ; Cynthia J. Burrows ; Sheila S. David
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：August 21, 2007
• 年：2007
• 卷：46
• 期：33
• 页码：9355 - 9365
• 全文大小：228K
• 年卷期：v.46,no.33(August 21, 2007)
• ISSN：1520-4995

文摘

Oxidation of guanine (G) and 8-oxoguanine (OG) with a wide variety of oxidants yields thehydantoin lesions, guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp). These two lesions havegarnered much recent attention due to their unusual structures and high mutagenic potential. We havepreviously shown that duplexes containing Gh and Sp are substrates for the base excision repair glycosylaseEscherichia coli Fpg (EcFpg). To evaluate the recognition features of these unusual lesions, binding andfootprinting experiments were performed using a glycosylase inactive variant, E3Q EcFpg, and 30 bpduplexes containing the embedded lesions. Surprisingly, E3Q EcFpg was found to bind significantly moretightly (~1000-fold) to duplexes containing Gh or Sp over the corresponding duplexes containing OG.This may be a consequence of the helix-destabilizing nature of the hydantoin lesions that facilitates theirrecognition within duplex DNA. Though DNA binding affinities of E3Q EcFpg with Gh- and Sp-containingduplexes were found to be similar to each other, hydroxyl radical footprinting using methidium-propyl-EDTA (MPE)-Fe(II) revealed subtle differences between binding of E3Q EcFpg to the two lesions. Mostnotably, in the presence of E3Q EcFpg, the Sp nucleotide (nt) is hyperreactive toward cleavage by MPE-Fe(II)-generated hydroxyl radicals, suggestive of the formation of an intercalation site for the MPE-Fe(II)reagent at the Sp nt. Interestingly, increasing the duplex length from 18 to 30 bp enhanced the excisionefficiency of Gh and Sp paired with C, G, or T by EcFpg such that these substrates are processed asefficiently as the signature substrate lesion, OG. Moreover, the base removal activity with these twolesions was more efficient than removal of OG when in a base pairing context opposite A. The highaffinity and efficient activity of EcFpg toward the hydantoin lesions suggest that EcFpg mediates repairof the lesions in vivo. Notably, the facile activity of EcFpg toward Gh and Sp in base pairing contextswith G and A, which are likely to be present after DNA replication, would be detrimental and enhancemutagenesis.