Unusual structural features of hydantoin lesions translate into efficient recognition by Escherichia coli Fpg.
Oxidation of guanine (G) and 8-oxoguanine (OG) with a wide variety of oxidants yields the hydantoin lesions, guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp). These two lesions have garnered much recent attention due to their unusual structures and high mutagenic potential. We have previously shown that duplexes containing Gh and Sp are substrates for the base excision repair glycosylase Escherichia coli Fpg (EcFpg). To evaluate the recognition features of these unusual lesions, binding and footprinting experiments were performed using a glycosylase inactive variant, E3Q EcFpg, and 30 bp duplexes containing the embedded lesions. Surprisingly, E3Q EcFpg was found to bind significantly more tightly ( approximately 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 their recognition within duplex DNA. Though DNA binding affinities of E3Q EcFpg with Gh- and Sp-containing duplexes 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. Most notably, 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 excision efficiency of Gh and Sp paired with C, G, or T by EcFpg such that these substrates are processed as efficiently as the signature substrate lesion, OG. Moreover, the base removal activity with these two lesions was more efficient than removal of OG when in a base pairing context opposite A. The high affinity and efficient activity of EcFpg toward the hydantoin lesions suggest that EcFpg mediates repair of the lesions in vivo. Notably, the facile activity of EcFpg toward Gh and Sp in base pairing contexts with G and A, which are likely to be present after DNA replication, would be detrimental and enhance mutagenesis.
Krishnamurthy
Nirmala
N
Department of Chemistry, University of Utah, 315 South, 1400 East, Salt Lake City, Utah 84112, USA.
Muller
James G
JG
Burrows
Cynthia J
CJ
David
Sheila S
SS
D020029:Base Pairing; D001483:Base Sequence; D002384:Catalysis; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004260:DNA Repair; D043382:DNA-Formamidopyrimidine Glycosylase / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D006146:Guanidines / Q000737:chemistry* / Q000378:metabolism; D006151:Guanosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D006827:Hydantoins / Q000737:chemistry* / Q000378:metabolism; D013141:Spiro Compounds / Q000737:chemistry* / Q000378:metabolism
D029968:Escherichia coli Proteins; D006146:Guanidines; D006827:Hydantoins; D013141:Spiro Compounds; C467338:guanidinohydantoin; C408822:spiroiminodihydantoin; D006151:Guanosine; D004247:DNA; D043382:DNA-Formamidopyrimidine Glycosylase; C468610:DNA-formamidopyrimidine glycosylase, E coli