DNA Lesion Recognition by the Bacterial Repair Enzyme MutM

Abstract

MutM is a bacterial DNA glycosylase that removes the mutagenic lesion 8-oxoguanine (oxoG) from duplex DNA. The means of oxoG recognition by MutM (also known as Fpg) is of fundamental interest, in light of the vast excess of normal guanine bases present in genomic DNA. The crystal structure of a recognition-competent but catalytically inactive version of MutM in complex with oxoG-containing DNA reveals the structural basis for recognition. MutM binds the oxoG nucleoside in the syn glycosidic configuration and distinguishes oxoG from guanine by reading out the protonation state of the N7 atom. The segment of MutM principally responsible for oxoG recognition is a flexible loop, suggesting that conformational mobility influences lesion recognition and catalysis. Furthermore, the structure of MutM in complex with DNA containing an alternative substrate, dihydrouracil, demonstrates how MutM is able to recognize lesions other than oxoG.The reactive byproducts of aerobic respiration oxidize DNA to generate a number of deleterious adducts, among which the most widely studied is 8-oxoguanine (oxoG). 1 Because oxoG mispairs with adenine during replication, this oxidative lesion is a source of G⅐C to T⅐A transversion mutations. Nearly all organisms possess enzymes that safeguard against the genotoxic effects of oxoG. The oxoG resistance pathway in bacteria, known as the "GO" system, comprises three components: MutT, MutY, and MutM (1-4). MutT hydrolyzes oxo-dGTP to oxodGMP and inorganic pyrophosphate so as to prevent de novo incorporation of oxoG into the genome during DNA replication. MutY initiates the repair of misreplicated oxoG⅐A pairs in DNA by catalyzing hydrolytic excision of the adenine base. MutM (also known as Fpg) is a bifunctional DNA glycosylase/lyase that catalyzes complete excision of oxoG lesion nucleosides when paired opposite C in DNA via a complex multistep reaction cascade. Most eukaryotes possess a GO system related to that in bacteria, with orthologous versions of MutT and MutY; however, in higher organisms, MutM is replaced by the functionally analogous but structurally unrelated enzyme Ogg1 (5, 6). Orthologs of MutM have been discovered in mammals, but these do not appear to be primarily involved in oxoG repair (7-10).Recent structural studies of MutM-DNA complexes have revealed the overall architecture of the protein-DNA complex. These structures have also provided insights into the general features of lesion presentation to the MutM active site, suggesting that the oxoG nucleoside is swiveled out of the DNA helix during repair, a theme common throughout the structural biology of base excision DNA repair. A segment of the protein near the active site is disordered in the DNA-bound MutM structures lacking an oxoG nucleobase, yet is ordered in the absence of DNA, thus leading to the tantalizing suggestion that induced fit might contribute to lesion base recognition. Understanding the structural determinants of lesion recognition by MutM has been hampered by the fact that none of the available high re...

The crystal structure of human endonuclease VIII-like 1 (NEIL1) reveals a zincless finger motif required for glycosylase activity

“…The difference, however, is not just due to the presence or absence of DNA, but seems to also hinge on the presence of a nucleobase lesion. The recent crystal structure of Bacillus stearothermophilus Fpg with lesion-containing DNA showed that this f lexible loop, which is positioned to recognize 8-oxoguanine, is ordered (see discussion below) (59). The structure overlay also revealed a 19-residue insertion in human NEIL1 (residues 204 -222), which comprises helix ␣F (Figs.…”

“…Recent crystal structures of B. stearothermophilus Fpg in complex with DNA containing either 8-oxoguanine or dihydrouracil revealed that the lesion is recognized by residues located in the ␣F-␤10 loop (for Fpg nomenclature, see ref. 59). It was further suggested that the mobility of the loop might play a part in lesion recognition and catalysis (59).…”

“…59). It was further suggested that the mobility of the loop might play a part in lesion recognition and catalysis (59). Interestingly, as mentioned above, in our structure of unliganded NEIL1, the corresponding segment is ordered similarly to what was described for uncomplexed TthFpg, although this region in NEIL1 is composed of a helix and a loop (helix ␣H and loop preceding it), rather than a loop.…”

Accelerated repair and reduced mutagenicity of oxidative DNA damage in human bladder cells expressing the E. coli FPG protein

“…As aforementioned, different substrate specificity and kinetics parameters characterize hOGG1 and FPG, the latter being able to release with similar excision kinetics 8-oxoG, FapyG and FapyA. 10 It has been further reported that FPG recognizes and excises oxidized pyrimidines from oligonucleotides with a single lesion only, 20,21 but no such repair activity could be detected on DNA substrates containing multiple lesions. 21,22 Hence, it is likely that the increased repair capacity of FPG-expressing cells is essentially limited to the enzyme physiological substrates i.e.…”

RNA editing changes the lesion specificity for the DNA repair enzyme NEIL1

“…In addition, while structural data for a complex of human NEIL1 with damaged DNA has not been reported, complexes of related repair glycosylases with DNA containing damaged bases have been structurally characterized (22,23). From an analysis of these structures, we realized the NEIL1 recoding site is located in the previously identified lesion recognition loop of this family of DNA repair enzymes ( Fig.…”