Enforced presentation of an extrahelical guanine to the lesion recognition pocket of human 8-oxoguanine glycosylase, hOGG1

A poorly understood aspect of DNA repair proteins is their ability to identify exceedingly rare sites of damage embedded in a large excess of nearly identical undamaged DNA, while catalyzing repair only at the damaged sites. Progress toward understanding this problem has been made by comparing the structures and biochemical behavior of these enzymes when they are presented with either a target lesion or a corresponding undamaged nucleobase. Trapping and analyzing such DNA-protein complexes is particularly difficult in the case of base extrusion DNA repair proteins because of the complexity of the repair reaction, which involves extrusion of the target base from DNA followed by its insertion into the active site where glycosidic bond cleavage is catalyzed. Here we report the structure of a human 8-oxoguanine (oxoG) DNA glycosylase, hOGG1, in which a normal guanine from DNA has been forcibly inserted into the enzyme active site. Although the interactions of the nucleobase with the active site are only subtly different for G versus oxoG, hOGG1 fails to catalyze excision of the normal nucleobase. This study demonstrates that even if hOGG1 mistakenly inserts a normal base into its active site, the enzyme can still reject it on the basis of catalytic incompatibility.     

Sequence-dependent structural variation in DNA undergoing intrahelical inspection by the DNA glycosylase MutM

MutM, a bacterial DNA-glycosylase, plays a critical role in maintaining genome integrity by catalyzing glycosidic bond cleavage of 8-oxoguanine (oxoG) lesions to initiate base excision DNA repair. The task faced by MutM of locating rare oxoG residues embedded in an overwhelming excess of undamaged bases is especially challenging given the close structural similarity between oxoG and its normal progenitor, guanine (G). MutM actively interrogates the DNA to detect the presence of an intrahelical, fully base-paired oxoG, whereupon the enzyme promotes extrusion of the target nucleobase from the DNA duplex and insertion into the extrahelical active site. Recent structural studies have begun to provide the first glimpse into the protein-DNA interactions that enable MutM to distinguish an intrahelical oxoG from G; however, these initial studies left open the important question of how MutM can recognize oxoG residues embedded in 16 different neighboring sequence contexts (considering only the 5'- and 3'-neighboring base pairs). In this study we set out to understand the manner and extent to which intrahelical lesion recognition varies as a function of the 5'-neighbor. Here we report a comprehensive, systematic structural analysis of the effect of the 5'-neighboring base pair on recognition of an intrahelical oxoG lesion. These structures reveal that MutM imposes the same extrusion-prone ("extrudogenic") backbone conformation on the oxoG lesion irrespective of its 5'-neighbor while leaving the rest of the DNA relatively free to adjust to the particular demands of individual sequences.     

Entrapment and Structure of an Extrahelical Guanine Attempting to Enter the Active Site of a Bacterial DNA Glycosylase, MutM

MutM, a bacterial DNA glycosylase, protects genome integrity by catalyzing glycosidic bond cleavage of 8-oxoguanine (oxoG) lesions, thereby initiating base excision DNA repair. The process of searching for and locating oxoG lesions is especially challenging, because of the close structural resemblance of oxoG to its million-fold more abundant progenitor, G. Extrusion of the target nucleobase from the DNA double helix to an extrahelical position is an essential step in lesion recognition and catalysis by MutM. Although the interactions between the extruded oxoG and the active site of MutM have been well characterized, little is known in structural detail regarding the interrogation of extruded normal DNA bases by MutM. Here we report the capture and structural elucidation of a complex in which MutM is attempting to present an undamaged G to its active site. The structure of this MutM-extrahelical G complex provides insights into the mechanism MutM employs to discriminate against extrahelical normal DNA bases and into the base extrusion process in general.     

Interaction features of adenine DNA glycosylase MutY from <Emphasis Type="Italic">E. coli</Emphasis> with DNA substrates

Kinetic characteristics of specific recognition of damaged base by the DNA glycosylase MutY in model DNA substrates, containing oxoG/A-, G/A-, oxoG/C- and F/G pairs in the central position, were investigated. Conformational changes of the MutY enzyme during the recognition of the damaged base in DNA have been recorded by the change in the fluorescence intensity of tryptophan residues using the stopped-flow technique in real time. DNA duplexes containing a fluorescein residue were used for the registration of DNA conformational changes. Analysis of the kinetic curves allowed us to determine the values of rate constants for the kinetic stages of the interaction. It was shown that nonspecific contacts between the DNA-binding site of the enzyme and the DNA duplex are formed at the first stage of the interaction. It was found that the discrimination of Gua and oxoGua bases occurs at the second stage of the MutY interaction with the DNA duplex. The data obtained for the oxoG/C-substrate showed that the recognition of the base located opposite oxoGua also occurs at this stage.     

Structural and Biochemical Analysis of DNA Helix Invasion by the Bacterial 8-Oxoguanine DNA Glycosylase MutM

MutM is a bacterial DNA glycosylase that serves as the first line of defense against the highly mutagenic 8-oxoguanine (oxoG) lesion, catalyzing glycosidic bond cleavage of oxoG to initiate base excision DNA repair. Previous work has shown that MutM actively interrogates DNA for the presence of an intrahelical oxoG lesion. This interrogation process involves significant buckling and bending of the DNA to promote extrusion of oxoG from the duplex. Structural snapshots have revealed several different highly conserved residues that are prominently inserted into the duplex in the vicinity of the target oxoG before and after base extrusion has occurred. However, the roles of these helix-invading residues during the lesion recognition and base extrusion process remain unclear. In this study, we set out to probe the function of residues Phe¹¹⁴ and Met⁷⁷ in oxoG recognition and repair. Here we report a detailed biochemical and structural characterization of MutM variants containing either a F114A or M77A mutation, both of which showed significant decreases in the efficiency of oxoG repair. These data reveal that Met⁷⁷ plays an important role in stabilizing the lesion-extruded conformation of the DNA. Phe¹¹⁴, on the other hand, appears to destabilize the intrahelical state of the oxoG lesion, primarily by buckling the target base pair. We report the observation of a completely unexpected interaction state, in which the target base pair is ruptured but remains fully intrahelical; this structure vividly illustrates the disruptive influence of MutM on the target base pair.     

Step-by-step mechanism of DNA damage recognition by human 8-oxoguanine DNA glycosylase

Background

Extensive structural studies of human DNA glycosylase hOGG1 have revealed essential conformational changes of the enzyme. However, at present there is little information about the time scale of the rearrangements of the protein structure as well as the dynamic behavior of individual amino acids.

Methods

Using pre-steady-state kinetic analysis with Trp and 2-aminopurine fluorescence detection the conformational dynamics of hOGG1 wild-type (WT) and mutants Y203W, Y203A, H270W, F45W, F319W and K249Q as well as DNA–substrates was examined.

Results

The roles of catalytically important amino acids F45, Y203, K249, H270, and F319 in the hOGG1 enzymatic pathway and their involvement in the step-by-step mechanism of oxidative DNA lesion recognition and catalysis were elucidated.

Conclusions

The results show that Tyr-203 participates in the initial steps of the lesion site recognition. The interaction of the His-270 residue with the oxoG base plays a key role in the insertion of the damaged base into the active site. Lys-249 participates not only in the catalytic stages but also in the processes of local duplex distortion and flipping out of the oxoG residue. Non-damaged DNA does not form a stable complex with hOGG1, although a complex with a flipped out guanine base can be formed transiently.

General significance

The kinetic data obtained in this study significantly improves our understanding of the molecular mechanism of lesion recognition by hOGG1.     

Separation-of-function mutants unravel the dual-reaction mode of human 8-oxoguanine DNA glycosylase

7,8-Dihydro-8-oxoguanine (8oxoG) is a major mutagenic base lesion formed when reactive oxygen species react with guanine in DNA. The human 8oxoG DNA glycosylase (hOgg1) recognizes and initiates repair of 8oxoG. hOgg1 is acknowledged as a bifunctional DNA glycosylase catalyzing removal of the damaged base followed by cleavage of the backbone of the intermediate abasic DNA (AP lyase/β-elimination). When acting on 8oxoG-containing DNA, these two steps in the hOgg1 catalysis are considered coupled, with Lys249 implicated as a key residue. However, several lines of evidence point to a concurrent and independent monofunctional hydrolysis of the N-glycosylic bond being the in?vivo relevant reaction mode of hOgg1. Here, we present biochemical and structural evidence for the monofunctional mode of hOgg1 by design of separation-of-function mutants. Asp268 is identified as the catalytic residue, while Lys249 appears critical for the specific recognition and final alignment of 8oxoG during the hydrolysis reaction.     

Structural characterization of human 8-oxoguanine DNA glycosylase variants bearing active site mutations

The human 8-oxoguanine DNA glycosylase (hOGG1) protein is responsible for initiating base excision DNA repair of the endogenous mutagen 8-oxoguanine. Like nearly all DNA glycosylases, hOGG1 extrudes its substrate from the DNA helix and inserts it into an extrahelical enzyme active site pocket lined with residues that participate in lesion recognition and catalysis. Structural analysis has been performed on mutant versions of hOGG1 having changes in catalytic residues but not on variants having altered 7,8-dihydro-8-oxoguanine (oxoG) contact residues. Here we report high resolution structural analysis of such recognition variants. We found that Ala substitution at residues that contact the phosphate 5' to the lesion (H270A mutation) and its Watson-Crick face (Q315A mutation) simply removed key functionality from the contact interface but otherwise had no effect on structure. Ala substitution at the only residue making an oxoG-specific contact (G42A mutation) introduced torsional stress into the DNA contact surface of hOGG1, but this was overcome by local interactions within the folded protein, indicating that this oxoG recognition motif is "hardwired." Introduction of a side chain intended to sterically obstruct the active site pocket (Q315F mutation) led to two different structures, one of which (Q315F(*149)) has the oxoG lesion in an exosite flanking the active site and the other of which (Q315F(*292)) has the oxoG inserted nearly completely into the lesion recognition pocket. The latter structure offers a view of the latest stage in the base extrusion pathway yet observed, and its lack of catalytic activity demonstrates that the transition state for displacement of the lesion base is geometrically demanding.     

How DNA Polymerase X Preferentially Accommodates Incoming dATP Opposite 8-Oxoguanine on the Template

The modified base 8-oxo-7,8-dihydro-2′-deoxyguanosine (oxoG) is a common DNA adduct produced by the oxidation of DNA by reactive oxygen species. Kinetic data reveal that DNA polymerase X (pol X) from the African swine fever virus incorporates adenine (dATP) opposite to oxoG with higher efficiency than the non-damaged G:C basepair. To help interpret the kinetic data, we perform molecular dynamics simulations of pol X/DNA complexes, in which the template base opposite to the incoming dNTP (dCTP, dATP, dGTP) is oxoG. Our results suggest that pol X accommodates the oxoG_syn:A mispair by sampling closed active conformations that mirror those observed in traditional Watson-Crick complexes. Moreover, for both the oxoG_syn:A and oxoG:C ternary complexes, conformational sampling of the polymerase follows previously described large subdomain movements, local residue motions, and active site reorganization. Interestingly, the oxoG_syn:A system exhibits superior active site geometry in comparison to the oxoG:C system. Simulations for the other mismatch basepair complexes reveal large protein subdomain movement for all systems, except for oxoG:G, which samples conformations close to the open state. In addition, active site geometry and basepairing of the template base with the incoming nucleotide, reveal distortions and misalignments that range from moderate (i.e., oxoG:A_syn) to extreme (i.e., oxoG_anti/syn:G). These results agree with the available kinetic data for pol X and provide structural insights regarding the mechanism by which this polymerase can accommodate incoming nucleotides opposite oxoG. Our simulations also support the notion that α-helix E is involved both in DNA binding and active site stabilization. Our proposed mechanism by which pol X can preferentially accommodate dATP opposite template oxoG further underscores the role that enzyme dynamics and conformational sampling operate in polymerase fidelity and function.     

Human DNA polymerase lambda is a proficient extender of primer ends paired to 7,8-dihydro-8-oxoguanine

Pol lambda is a DNA repair enzyme with a high affinity for dNTPs, an intrinsic dRP lyase activity, a BRCT domain involved in interactions with NHEJ factors, and also capable to interact with the PCNA processivity factor. Based on this potential, Pol lambda could play a role in BER, V(D)J recombination, NHEJ and TLS. Here we show that human Pol lambda uses a templating 7,8-dihydro-8-oxoguanine (8oxoG) base, a common mutagenic form of oxidative damage, as efficiently as an undamaged dG, but giving rise to the alternative insertion of either dAMP or dCMP. However, Pol lambda strongly discriminated against the extension of the mutagenic 8oxoG:dAMP pair. Conversely, Pol lambda readily extended the non-mutagenic 8oxoG:dCMP pair with an efficiency that was even higher than that displayed on undamaged dG:dCMP pair. A similar capacity for non-mutagenic extension was also shown to occur in the case of O6-methylguanine (m6G), a mutagenic and cytotoxic DNA adduct. A comparison of these novel properties of human Pol lambda with those of other DNA polymerases involved in TLS will be discussed. Interestingly, when double-strand breaks are associated to base damage, modifications as 8oxoG could be eventually part of the synapsis required to join ends, and therefore, the capacity of Pol lambda either to insert opposite 8oxoG or to extend correct base pairs containing such a damage could be beneficial for its role in NHEJ.     

Evidence that MutY is a monofunctional glycosylase capable of forming a covalent Schiff base intermediate with substrate DNA.

The Escherichia coli adenine glycosylase MutY is involved in the repair of 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG):A and G:A mispairs in DNA. DNA strand cleavage via beta-elimination (beta-lyase) activity coupled with MutY's removal of misincorporated adenine bases was sought using both qualitative and quantitative methods. The qualitative assays demonstrate formation of a Schiff base intermediate which is characteristic of DNA glycosylases catalyzing a concomitant beta-lyase reaction. Borohydride reduction of the Schiff base results in the formation of a covalent DNA-MutY adduct which is easily detected in SDS-PAGE experiments. However, quantitative activity assays which monitor DNA strand scission accompanying base release suggest MutY behaves as a simple monofunctional glycosylase. Treatment with base effects DNA strand cleavage at apurinic/apyrimidinic (AP) sites arising via simple glycosylase activity. The amount of cleaved DNA in MutY reactions treated with base is much greater than that in non-base treated reactions, indicating that AP site generation by MutY is not associated with a concomitant beta-lyase step. As standards, identical assays were performed with a known monofunctional enzyme (uracil DNA glycosylase) and a known bifunctional glycosylase/lyase (FPG), the results of which were used in comparison with those of the MutY experiments. The apparent inconsistency between the data obtained for MutY by the qualitative and quantitative methods underscores the current debate surrounding the catalytic activity of this enzyme, and a detailed explanation of this controversy is proposed. The work presented here lays ground for the identification of specific active site residues responsible for the chemical mechanism of MutY enzyme catalysis.     

The influence of combined Fpg- and MutY-deficiency on the spontaneous and gamma-radiation-induced mutation spectrum in the lacZalpha gene of m13mp10

One of the most predominating oxidative DNA damages, both spontaneously formed and after gamma-radiation is 7, 8-dihydro-8-oxoguanine (8oxoG). This 8oxoG is a mutagenic lesion because it can mispair with adenine instead of the correct cytosine leading to G:C to T:A transversions. In Escherichia coli (E. Coli) base excision repair (BER) is one of the most important repair systems for the repair of 8oxoG and other oxidative DNA damage. An important part of BER in E. coli is the so-called GO system which consists of three repair enzymes, MutM (Fpg), MutY and MutT which are all involved in repair of 8oxoG or 8oxoG mispairs. The aim of this study is to determine the effect of combined Fpg- and MutY-deficiency on the spontaneous and gamma-radiation-induced mutation spectrum of the lacZalpha gene. For that purpose, non-irradiated or gamma-irradiated double-stranded (ds) M13mp10 DNA, with the lacZalpha gene inserted as mutational target sequence was transfected into an E. coli strain which is deficient in both Fpg and MutY (BH1040). The resulting mutation spectra were compared with the mutation spectra of a fpg(-) E. coli strain (BH410) and a wild type E. coli strain (JM105) which were determined in an earlier study. The results of the present study indicate that combined Fpg- and MutY-deficiency induces a large increase in G:C to T:A transversions in both the spontaneous and gamma-radiation-induced mutation spectra of BH1040 (fpg(-)mutY(-)) as compared to the fpg(-) and the wild type strain. Besides the increased levels of G:C to T:A transversions, there is also an increase in G:C to C:G transversions and frameshift mutations in both the spontaneous and gamma-radiation-induced mutation spectra of BH1040 (fpg(-)mutY(-)).     

How DNA Polymerase X Preferentially Accommodates Incoming dATP Opposite 8-Oxoguanine on the Template

The modified base 8-oxo-7,8-dihydro-2′-deoxyguanosine (oxoG) is a common DNA adduct produced by the oxidation of DNA by reactive oxygen species. Kinetic data reveal that DNA polymerase X (pol X) from the African swine fever virus incorporates adenine (dATP) opposite to oxoG with higher efficiency than the non-damaged G:C basepair. To help interpret the kinetic data, we perform molecular dynamics simulations of pol X/DNA complexes, in which the template base opposite to the incoming dNTP (dCTP, dATP, dGTP) is oxoG. Our results suggest that pol X accommodates the oxoG_syn:A mispair by sampling closed active conformations that mirror those observed in traditional Watson-Crick complexes. Moreover, for both the oxoG_syn:A and oxoG:C ternary complexes, conformational sampling of the polymerase follows previously described large subdomain movements, local residue motions, and active site reorganization. Interestingly, the oxoG_syn:A system exhibits superior active site geometry in comparison to the oxoG:C system. Simulations for the other mismatch basepair complexes reveal large protein subdomain movement for all systems, except for oxoG:G, which samples conformations close to the open state. In addition, active site geometry and basepairing of the template base with the incoming nucleotide, reveal distortions and misalignments that range from moderate (i.e., oxoG:A_syn) to extreme (i.e., oxoG_anti/syn:G). These results agree with the available kinetic data for pol X and provide structural insights regarding the mechanism by which this polymerase can accommodate incoming nucleotides opposite oxoG. Our simulations also support the notion that α-helix E is involved both in DNA binding and active site stabilization. Our proposed mechanism by which pol X can preferentially accommodate dATP opposite template oxoG further underscores the role that enzyme dynamics and conformational sampling operate in polymerase fidelity and function.     

Effect O6‐guanine alkylation on DNA flexibility studied by comparative molecular dynamics simulations

Alkylation of guanine at the O6 atom is a highly mutagenic DNA lesion because it alters the coding specificity of the base causing G:C to A:T transversion mutations. Specific DNA repair enzymes, e.g. O⁶‐alkylguanin‐DNA‐Transferases (AGT), recognize and repair such damage after looping out the damaged base to transfer it into the enzyme active site. The exact mechanism how the repair enzyme identifies a damaged site within a large surplus of undamaged DNA is not fully understood. The O⁶‐alkylation of guanine may change the deformability of DNA which may facilitate the initial binding of a repair enzyme at the damaged site. In order to characterize the effect of O⁶‐methyl‐guanine (O⁶‐MeG) containing base pairs on the DNA deformability extensive comparative molecular dynamics (MD) simulations on duplex DNA with central G:C, O⁶‐MeG:C or O⁶‐MeG:T base pairs were performed. The simulations indicate significant differences in the helical deformability due to the presence of O⁶‐MeG compared to regular undamaged DNA. This includes enhanced base pair opening, shear and stagger motions and alterations in the backbone fine structure caused in part by transient rupture of the base pairing at the damaged site and transient insertion of water molecules. It is likely that the increased opening motions of O⁶‐MeG:C or O⁶‐MeG:T base pairs play a decisive role for the induced fit recognition or for the looping out of the damaged base by repair enzymes. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 23–32, 2015.     

Characterization of DNA with an 8-oxoguanine modification

The oxidation of DNA resulting from reactive oxygen species generated during aerobic respiration is a major cause of genetic damage that, if not repaired, can lead to mutations and potentially an increase in the incidence of cancer and aging. A major oxidation product generated in cells is 8-oxoguanine (oxoG), which is removed from the nucleotide pool by the enzymatic hydrolysis of 8-oxo-2'-deoxyguanosine triphosphate and from genomic DNA by 8-oxoguanine-DNA glycosylase. Finding and repairing oxoG in the midst of a large excess of unmodified DNA requires a combination of rapid scanning of the DNA for the lesion followed by specific excision of the damaged base. The repair of oxoG involves flipping the lesion out of the DNA stack and into the active site of the 8-oxoguanine-DNA glycosylase. This would suggest that thermodynamic stability, in terms of the rate for local denaturation, could play a role in lesion recognition. While prior X-ray crystal and NMR structures show that DNA with oxoG lesions appears virtually identical to the corresponding unmodified duplex, thermodynamic studies indicate that oxoG has a destabilizing influence. Our studies show that oxoG destabilizes DNA (ΔΔG of 2-8 kcal mol(-1) over a 16-116 mM NaCl range) due to a significant reduction in the enthalpy term. The presence of oxoG has a profound effect on the level and nature of DNA hydration indicating that the environment around an oxoG?C is fundamentally different than that found at G?C. The temperature-dependent imino proton NMR spectrum of oxoG modified DNA confirms the destabilization of the oxoG?C pairing and those base pairs that are 5' of the lesion. The instability of the oxoG modification is attributed to changes in the hydrophilicity of the base and its impact on major groove cation binding.     

The substrate specificity of MutY for hyperoxidized guanine lesions in vivo

The DNA damage product 7,8-dihydro-8-oxo-2'-deoxyguanine (8-oxoG) is a commonly used biomarker of oxidative stress. The mutagenic potential of this DNA lesion is mitigated in Escherichia coli by multiple enzymes. One of these enzymes, MutY, excises an A mispaired with 8-oxoG as part of the process to restore the original G:C base pair. However, numerous studies have shown that 8-oxoG is chemically labile toward further oxidation. Here, we examine the activity of MutY on the 8-oxoG oxidation products guanidinohydantoin (Gh), two diastereomers of spiroiminodihydantoin (Sp1 and Sp2), oxaluric acid (Oa), and urea (Ur). Single-stranded viral genomes containing a site-specific lesion were constructed and replicated in E. coli that are either proficient in DNA repair or that lack MutY. These lesions were found previously to be potently mutagenic in repair competent bacteria, and we report here that these 8-oxoG-derived lesions are equally miscoding when replicated in E. coli lacking MutY; no significant change in mutation identity or frequency is observed. Interestingly, however, in the presence of MutY, Sp1 and Sp2 are more toxic than in cells lacking this repair enzyme.     

Active destabilization of base pairs by a DNA glycosylase wedge initiates damage recognition

Formamidopyrimidine-DNA glycosylase (Fpg) excises 8-oxoguanine (oxoG) from DNA but ignores normal guanine. We combined molecular dynamics simulation and stopped-flow kinetics with fluorescence detection to track the events in the recognition of oxoG by Fpg and its mutants with a key phenylalanine residue, which intercalates next to the damaged base, changed to either alanine (F110A) or fluorescent reporter tryptophan (F110W). Guanine was sampled by Fpg, as evident from the F110W stopped-flow traces, but less extensively than oxoG. The wedgeless F110A enzyme could bend DNA but failed to proceed further in oxoG recognition. Modeling of the base eversion with energy decomposition suggested that the wedge destabilizes the intrahelical base primarily through buckling both surrounding base pairs. Replacement of oxoG with abasic (AP) site rescued the activity, and calculations suggested that wedge insertion is not required for AP site destabilization and eversion. Our results suggest that Fpg, and possibly other DNA glycosylases, convert part of the binding energy into active destabilization of their substrates, using the energy differences between normal and damaged bases for fast substrate discrimination.     

A dimeric mechanism for contextual target recognition by MutY glycosylase

MutY, an adenine glycosylase, initiates the critical repair of an adenine:8-oxo-guanine base pair in DNA arising from polymerase error at the oxidatively damaged guanine. Here we demonstrate for the first time, using presteady-state active site titrations, that MutY assembles into a dimer upon binding substrate DNA and that the dimer is the functionally active form of the enzyme. Additionally, we observed allosteric inhibition of glycosylase activity in the dimer by the concurrent binding of two lesion mispairs. Active site titration results were independently verified by gel mobility shift assays and quantitative DNA footprint titrations. A model is proposed for the potential functional role of the observed polysteric and allosteric regulation in recruiting and coordinating interactions with the methyl-directed mismatch repair system.     

极端嗜热古菌8oxoG DNA糖苷酶的研究进展

7,8二氢-8-氧鸟嘌呤(7,8-dihydro-8-oxoguanine,8oxoG)是一种常见的DNA损伤碱基.由于8oxoG能够与腺嘌呤配对,在DNA中的8oxoG被修复之前进行复制,DNA将会产生GC→TA的突变,从而造成基因组的不稳定.目前,碱基切除修复(Base excision repair,BER)是修...     

DNA ligation catalyzed by human topoisomerase II alpha

The DNA ligation reaction of topoisomerase II is essential for genomic integrity. However, it has been impossible to examine many fundamental aspects of this reaction because ligation assays historically required the enzyme to cleave a DNA substrate before sealing the nucleic acid break. Recently, a cleavage-independent DNA ligation assay was developed for human topoisomerase IIalpha [Bromberg, K. D., Hendricks, C., Burgin, A. B., and Osheroff, N. (2002) J. Biol. Chem. 277, 31201-31206]. This assay overcomes the requirement for DNA cleavage by monitoring the ability of the enzyme to ligate a nicked oligonucleotide in which the 5'-terminal phosphate at the nick has been activated by covalent attachment to the tyrosine mimic, p-nitrophenol. The cleavage-independent ligation assay was used to more fully characterize the DNA ligation activity of human topoisomerase IIalpha. Results suggest that the active site tyrosine contributes little to the catalysis of DNA ligation beyond its primary role as an activating/leaving group. Although arginine 804 (the residue immediately N-terminal to the active site tyrosine) has been proposed to help anchor the 5'-DNA terminus during cleavage, conversion of this residue to alanine had only a modest effect on DNA ligation. Thus, it appears that arginine 804 does not play an essential role in DNA strand joining. In contrast, disruption of base pairing at the 5'-DNA terminus abrogated DNA ligation in the absence of a covalent enzyme-DNA bond. Therefore, it is proposed that base pairing represents a secondary mechanism for aligning the 5'-DNA termini for ligation. Finally, the human enzyme appears to ligate the two scissile bonds of a cleavage site in a nonconcerted fashion.