Unnatural substrates reveal the importance of 8-oxoguanine for in vivo mismatch repair by MutY

Abstract

Escherchia coli MutY plays an important role in preventing mutations associated with the oxidative lesion 7,8-dihydro-8-oxo-2′-deoxyguanosine (OG) in DNA by excising adenines from OG:A mismatches as the first step of base excision repair. To determine the importance of specific steps in the base pair recognition and base removal process of MutY, we have evaluated the effects of modifications of the OG:A substrate on the kinetics of base removal, mismatch affinity and repair to G:C in an Escherchia coli-based assay. Surprisingly, adenine modification was tolerated in the cellular assay, while modification of OG results in minimal cellular repair. High affinity for the mismatch and efficient base removal require the presence of OG. Taken together, these results suggest that the presence of OG is a critical feature for MutY to locate OG:A mismatches and select the appropriate adenines for excision to initiate repair in vivo prior to replication.The mismatch repair (MMR) pathway in E. coli relies on methylation at a GATC sequence to direct the repair machinery to remove the mismatched base on the newly synthesized strand. 1 However, almost two decades ago, an activity was detected in E. coli cell extracts that restored G:A mismatches to G:C matches and was insensitive to the methylation state of the template strand. [2][3][4] Concurrently, a mutator locus in E. coli (mutY) was identified that generated G:C → T:A transversion mutations. 5 It was later determined that the mutY gene product is an adenine glycosylase capable of removing adenines from G:A mismatches as the first step in base excision repair (BER). [6][7][8][9][10] The subsequent activity of downstream BER pathway enzymes, e.g. the AP endonuclease, deoxyribophosphate lyase, polymerase and ligase, restores the G:C base pair in a methylation-independent fashion. 11 MutY was also found to participate in the prevention of mutations caused by 7,8-dihydro-8-oxo-2′-deoxyguanosine (OG, 1) by removal of adenine from OG:A mismatches. 10,12,13,14 Polymerase misinsertion of dAMP opposite OG creates OG:A mismatches that can lead to formation of T:A base pairs upon a second round of replication. 12 Recently, MutY has been in the spotlight due to the correlation between inherited biallelic mutations in the gene encoding the human homologue of MutY (MUTYH) and colorectal cancer. 10,15,16 *Author to whom correspondence should be addressed. Email: david@chem.ucdavis.edu,. # These authors contributed equally to this manuscript. We have previously examined the features of mismatched substrates that are required for efficient lesion recognition and adenine excision by MutY using pre-steady state and singleturnover kinetics. [17][18][19] Pre-steady state experiments revealed that MutY has a high affinity for the product such that release of the DNA product is rate-limiting. 17,19,20 Moreover, the identity of the base opposite A greatly affects both the rate of product release as well as the intrinsic rate of adenine removal determined under single-turnover conditions....

Gas-Phase Studies of Substrates for the DNA Mismatch Repair Enzyme MutY

“…Q and Z1 are more easily cleaved than would be predicted by the acidities in Figure 14, but both have one feature in common: a nitrogen at N3, which has been proposed to be important for MutY excision. 14,16,25,26 …”

Atomic substitution reveals the structural basis for substrate adenine recognition and removal by adenine DNA glycosylase

“…3D, not shown in Fig. 5A), but we ruled out this second water as the catalytic nucleophile, both because of its poor positioning but also because E. coli MutY efficiently processes the substrate analog 3-deazaadenine (40). The involvement of Glu-43 in protonating the substrate adenine and Asp-144 in deprotonating the catalytic water had been proposed earlier by Tainer and colleagues on the basis of the structure of a mutant E. coli MutY bound to adenine (corresponding residues Glu-37 and Asp-138) (12).…”

Redox signaling between DNA repair proteins for efficient lesion detection

“…Location of damaged bases in the genome is likely the rate-limiting step in BER within the cell (6). Current models for genome scanning to detect lesions involve protein sliding along the DNA, squeezing the backbone, slipping bases out to allow for interrogation, or finding transiently opened sites (7,8).…”