Maintaining the chemical integrity of DNA in the face of assault by oxidizing agents is a constant challenge for living organisms. Base-excision repair has an important role in preventing mutations associated with a common product of oxidative damage to DNA, 8-oxoguanine. Recent structural studies have shown that 8-oxoguanine DNA glycosylases use an intricate series of steps to locate and excise 8-oxoguanine lesions efficiently against a high background of undamaged bases. The importance of preventing mutations associated with 8-oxoguanine is shown by a direct association between defects in the DNA glycosylase MUTYH and colorectal cancer. The properties of other guanine oxidation products and the associated DNA glycosylases that remove them are now also being revealed.
MutY and endonuclease III, two DNA glycosylases from Escherichia coli, and AfUDG, a uracil DNA glycosylase from Archeoglobus fulgidus, are all base excision repair enzymes that contain the [4Fe-4S](2+) cofactor. Here we demonstrate that, when bound to DNA, these repair enzymes become redox-active; binding to DNA shifts the redox potential of the [4Fe-4S](3+/2+) couple to the range characteristic of high-potential iron proteins and activates the proteins toward oxidation. Electrochemistry on DNA-modified electrodes reveals potentials for Endo III and AfUDG of 58 and 95 mV versus NHE, respectively, comparable to 90 mV for MutY bound to DNA. In the absence of DNA modification of the electrode, no redox activity can be detected, and on electrodes modified with DNA containing an abasic site, the redox signals are dramatically attenuated; these observations show that the DNA base pair stack mediates electron transfer to the protein, and the potentials determined are for the DNA-bound protein. In EPR experiments at 10 K, redox activation upon DNA binding is also evident to yield the oxidized [4Fe-4S](3+) cluster and the partially degraded [3Fe-4S](1+) cluster. EPR signals at g = 2.02 and 1.99 for MutY and g = 2.03 and 2.01 for Endo III are seen upon oxidation of these proteins by Co(phen)(3)(3+) in the presence of DNA and are characteristic of [3Fe-4S](1+) clusters, while oxidation of AfUDG bound to DNA yields EPR signals at g = 2.13, 2.04, and 2.02, indicative of both [4Fe-4S](3+) and [3Fe-4S](1+) clusters. On the basis of this DNA-dependent redox activity, we propose a model for the rapid detection of DNA lesions using DNA-mediated electron transfer among these repair enzymes; redox activation upon DNA binding and charge transfer through well-matched DNA to an alternate bound repair protein can lead to the rapid redistribution of proteins onto genome sites in the vicinity of DNA lesions. This redox activation furthermore establishes a functional role for the ubiquitous [4Fe-4S] clusters in DNA repair enzymes that involves redox chemistry and provides a means to consider DNA-mediated signaling within the cell.
MutY adenine glycosylases prevent DNA mutations by excising adenine from promutagenic 8-oxo-7,8-dihydroguanine (OG):A mismatches. Here, we describe structural features of the MutY active site bound to an azaribose transition state analog which indicate a catalytic role for Tyr126 and approach of the water nucleophile on the same side as the departing adenine base. The idea that Tyr126 participates in catalysis, recently predicted by modeling calculations, is strongly supported by mutagenesis and by seeing close contact between the hydroxyl group of this residue and the azaribose moiety of the transition state analog. NMR analysis of MutY methanolysis products corroborates a mechanism for adenine removal with retention of stereochemistry. Based on these results, we propose a revised mechanism for MutY that involves two nucleophilic displacement steps akin to the mechanisms accepted for ‘retaining’ O-glycosidases. This new-for-MutY yet familiar mechanism may also be operative in related base excision repair glycosylases and provides a critical framework for analysis of human MutY (MUTYH) variants associated with inherited colorectal cancer.
DNA charge transport (CT) chemistry provides a route to carry out oxidative DNA damage from a distance in a reaction that is sensitive to DNA mismatches and lesions. Here, DNA-mediated CT also leads to oxidation of a DNA-bound base excision repair enzyme, MutY. DNA-bound Ru(III), generated through a flash͞ quench technique, is found to promote oxidation of the 1؉ clusters. In ruthenium-tethered DNA assemblies, oxidative damage to the 5-G of a 5-GG-3 doublet is generated from a distance but this irreversible damage is inhibited by MutY and instead EPR experiments reveal cluster oxidation. With rutheniumtethered assemblies containing duplex versus single-stranded regions, MutY oxidation is found to be mediated by the DNA duplex, with guanine radical as an intermediate oxidant; guanine radical formation facilitates MutY oxidation. A model is proposed for the redox activation of DNA repair proteins through DNA CT, with guanine radicals, the first product under oxidative stress, in oxidizing the DNA-bound repair proteins, providing the signal to stimulate DNA repair.electron transfer ͉ iron-sulfur cluster ͉ oxidative DNA damage D NA-mediated charge transport (CT) from a distance to generate oxidative damage was first demonstrated in an assembly containing a tethered metallointercalator (1). In this assembly, photoinduced oxidative damage of the 5Ј-G of 5Ј-GG-3Ј sites was observed; this damage pattern has since become the hallmark of DNA CT chemistry, and long-range oxidative damage has been confirmed by using a variety of pendant oxidants (2-6). Long-range oxidative DNA damage has been demonstrated over a distance of at least 200 Å (7, 8). Indeed, DNA either packaged in nucleosome core particles (9) or inside the cell nucleus (10) has been found to be susceptible to long-range oxidative damage. Chemically well defined assemblies, consisting of DNA duplexes with covalently bound oxidants, have been particularly useful in establishing the sensitivity of DNA CT to base-stacking perturbation (11-16). Recently, analogous studies probing long-range reductive chemistry on DNA has been probed both in solution (17-20) and on DNAmodified surfaces (14,15,21). As with oxidation chemistry, these reactions show only small variations in rate with distance but are remarkably sensitive to perturbations in the intervening base pair stack. Mechanistic descriptions for DNA CT focused first on a mixture of hopping and tunneling. A phonon-assisted polaron model has also been put forth (22). Studies as a function of temperature have shown the CT process to be gated by base pair dynamics; in fact, base pair motions are required for CT (23, 24).We have therefore described DNA CT in the context of transport among delocalized DNA domains formed and dissolved based on sequence-dependent DNA dynamics.Given the exquisite sensitivity of DNA CT to DNA lesions and mismatches, we have recently explored a possible role for DNA CT in repair. We demonstrated that redox activity required DNA binding for MutY (25), a base excision repair (BER) enzyme fr...
Cellular replication forks are powered by ring-shaped, hexameric helicases that encircle and unwind DNA. To better understand the molecular mechanisms and control of these enzymes, we used multiple methods to investigate the bacterial replicative helicase, DnaB. A 3.3 Å crystal structure of Aquifex aeolicus DnaB complexed with nucleotide reveals a new conformational state for this motor protein. Electron microscopy and small angle X-ray scattering studies confirm the state seen crystallographically, showing that the DnaB ATPase domains and an associated N-terminal collar transition between two physical states in a nucleotide-dependent manner. Mutant helicases locked in either collar state are active, but display different capacities to support critical activities such as duplex translocation and primase-dependent RNA synthesis. Our findings establish the DnaB collar as an auto-regulatory hub that controls the ability of the helicase to transition between different functional states in response to nucleotide and both replication initiation and elongation factors.
Summary Dedicated AAA+ ATPases help deposit hexameric ring-shaped helicases onto DNA to promote replication in cellular organisms. To understand how loading occurs, we used negative-stain electron microscopy and small-angle X-ray scattering to determine the ATP-bound structure of the intact E. coli DnaB•DnaC helicase/loader complex. The 480 kDa dodecamer forms a three-tiered assembly, in which DnaC adopts a spiral configuration that remodels N-terminal scaffolding and C-terminal motor regions of DnaB to produce a clear break in the helicase ring. Surprisingly, DnaC’s AAA+ fold is dispensable for ring remodeling, as the isolated helicase-binding domain of DnaC can both load DnaB onto DNA and increase the efficiency by which the helicase acts on substrates in vitro. Our data demonstrate that DnaC opens DnaB by a mechanism akin to that of polymerase clamp loaders, and indicate that bacterial replicative helicases, like their eukaryotic counterparts, possess auto-regulatory elements that influence how the hexameric motor domains are loaded onto and unwind DNA.
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....
Despite a low copy number within the cell, base excision repair (BER) enzymes readily detect DNA base lesions and mismatches. These enzymes also contain [Fe 4S4] clusters, yet a redox role for these iron cofactors had been unclear. Here, we provide evidence that BER proteins may use DNA-mediated redox chemistry as part of a signaling mechanism to detect base lesions. By using chemically modified bases, we show electron trapping on DNA in solution with bound BER enzymes by electron paramagnetic resonance (EPR) spectroscopy. We demonstrate electron transfer from two BER proteins, Endonuclease III (EndoIII) and MutY, to modified bases in DNA containing oxidized nitroxyl radical EPR probes. Electron trapping requires that the modified base is coupled to the DNA -stack, and trapping efficiency is increased when a noncleavable MutY substrate analogue is located distally to the trap. These results are consistent with DNA binding leading to the activation of the repair proteins toward oxidation. Significantly, these results support a mechanism for DNA repair that involves DNA-mediated charge transport.base excision repair ͉ DNA charge transport ͉ iron sulfur clusters O ur laboratory has carried out a range of studies to probe and apply DNA-mediated charge transport (CT) chemistry (1). Oxidative damage to DNA from a distance through DNA CT was first demonstrated in an assembly containing a tethered metallointercalator (2). Since that time, photoinduced oxidative damage of the 5Ј-G of 5Ј-GG-3Ј sites from a distance has been observed in assemblies using various pendant oxidants (3-5). Indeed, long-range oxidative DNA damage has been demonstrated over a distance of at least 200 Å (6, 7). Besides the shallow distance dependence in reactions by DNA CT, another characteristic of this chemistry has been its exquisite sensitivity to perturbations in base pair structure. CT through DNA is inhibited by intervening DNA mismatches and bulges as well as by DNA-binding proteins that interfere with base pair stacking (8)(9)(10)(11)(12). Recently, studies probing long-range reductive chemistry on DNA also have been investigated both in solution (13-15) and on DNA-modified surfaces (12, 16). As with oxidation chemistry, these reactions show only small variations in rate with distance but are sensitive to perturbations in the intervening base pair stack.Given the unique characteristics of DNA CT chemistry, we are interested in whether DNA CT might be important physiologically. Based on the exquisite sensitivity of DNA CT to base pair lesions and mismatches, we considered in particular that DNA CT might be advantageous with respect to DNA repair. Base excision repair (BER) enzymes are exceedingly efficient in detecting DNA base lesions and mismatches, yet an understanding of that process has been elusive (17 2ϩ clusters are well separated from the enzyme active site and do not appear to participate in the glycoslyase reaction, yet they are essential for overall repair activity. Crystal structures in the absence and presence of DNA, moreov...
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