scholarly journals Catalytic mechanism of the mismatch-specific DNA glycosylase methyl-CpG-binding domain 4

2020 ◽  
Vol 477 (9) ◽  
pp. 1601-1612
Author(s):  
Hala Ouzon-Shubeita ◽  
Hunmin Jung ◽  
Michelle H. Lee ◽  
Myong-Chul Koag ◽  
Seongmin Lee
Keyword(s):  
Catalytic Mechanism ◽  
Bond Cleavage ◽  
Binding Domain ◽  
Base Catalysis ◽  
Dna Glycosylase ◽  
Dna Glycosylases ◽  
Base Pairs ◽  
Mismatched Dna ◽  
Ap Site ◽  
Domain 4

Thymine:guanine base pairs are major promutagenic mismatches occurring in DNA metabolism. If left unrepaired, these mispairs can cause C to T transition mutations. In humans, T:G mismatches are repaired in part by mismatch-specific DNA glycosylases such as methyl-CpG-binding domain 4 (hMBD4) and thymine-DNA glycosylase. Unlike lesion-specific DNA glycosylases, T:G-mismatch-specific DNA glycosylases specifically recognize both bases of the mismatch and remove the thymine but only from mispairs with guanine. Despite the advances in biochemical and structural characterizations of hMBD4, the catalytic mechanism of hMBD4 remains elusive. Herein, we report two structures of hMBD4 processing T:G-mismatched DNA. A high-resolution crystal structure of Asp560Asn hMBD4-T:G complex suggests that hMBD4-mediated glycosidic bond cleavage occurs via a general base catalysis mechanism assisted by Asp560. A structure of wild-type hMBD4 encountering T:G-containing DNA shows the generation of an apurinic/apyrimidinic (AP) site bearing the C1′-(S)-OH. The inversion of the stereochemistry at the C1′ of the AP-site indicates that a nucleophilic water molecule approaches from the back of the thymine substrate, suggesting a bimolecular displacement mechanism (SN2) for hMBD4-catalyzed thymine excision. The AP-site is stabilized by an extensive hydrogen bond network in the MBD4 catalytic site, highlighting the role of MBD4 in protecting the genotoxic AP-site.

scholarly journals Insights into the substrate discrimination mechanisms of methyl-CpG-binding domain 4

2021 ◽  
Author(s):  
Hala Ouzon-Shubeita ◽  
Lillian Feigang Schmaltz ◽  
Seongmin Lee
Keyword(s):  
Carbonyl Oxygen ◽  
Binding Domain ◽  
Dna Glycosylase ◽  
Side Chain ◽  
Dna Glycosylases ◽  
Base Pairs ◽  
Thymine Dna Glycosylase ◽  
Mismatched Dna ◽  
Domain 4

G:T mismatches, the major mispairs generated during DNA metabolism, are repaired in part by mismatch-specific DNA glycosylases such as methyl-CpG-binding domain 4 (MBD4) and thymine DNA glycosylase (TDG). Mismatch-specific DNA glycosylases must discriminate the mismatches against million-fold excess correct base pairs. MBD4 efficiently removes thymine opposite guanine but not opposite adenine. Previous studies have revealed that the substrate thymine is flipped out and enters the catalytic site of the enzyme, while the estranged guanine is stabilized by Arg468 of MBD4. To gain further insights into mismatch discrimination mechanism of MBD4, we assessed the glycosylase activity of MBD4 toward various base pairs. In addition, we determined a crystal structure of MBD4 bound to T:O6-methylguanine-containing DNA, which suggests the O6 and N2 of purine and the O4 of pyrimidine are required to be a substrate for MBD4. To understand the role of the Arg468 finger in catalysis, we evaluated the glycosylase activity of MBD4 mutants, which revealed the guanidinium moiety of Arg468 may play an important role in catalysis. D560N/R468K MBD4 bound to T:G mismatched DNA shows that the side chain amine moiety of the Lys stabilizes the flipped-out thymine by a water-mediated phosphate pinching, while the backbone carbonyl oxygen of the Lys engages in hydrogen bonds with N2 of the estranged guanine. Comparison of various DNA glycosylase structures implies the guanidinium and amine moieties of Arg and Lys, respectively, may involve in discriminating between substrate mismatches and nonsubstrate base pairs.

scholarly journals DNA glycosylases in the base excision repair of DNA

1997 ◽  
Vol 325 (1) ◽  
pp. 1-16 ◽  
Author(s):  
Hans E. KROKAN ◽  
Rune STANDAL ◽  
Geir SLUPPHAUG
Keyword(s):  
Base Excision Repair ◽  
Active Sites ◽  
Catalytic Mechanism ◽  
Excision Repair ◽  
Bacteriophage T4 ◽  
Pyrimidine Dimer ◽  
Dna Glycosylases ◽  
Wide Range ◽  
Base Excision ◽  
Ap Site

A wide range of cytotoxic and mutagenic DNA bases are removed by different DNA glycosylases, which initiate the base excision repair pathway. DNA glycosylases cleave the N-glycosylic bond between the target base and deoxyribose, thus releasing a free base and leaving an apurinic/apyrimidinic (AP) site. In addition, several DNA glycosylases are bifunctional, since they also display a lyase activity that cleaves the phosphodiester backbone 3′ to the AP site generated by the glycosylase activity. Structural data and sequence comparisons have identified common features among many of the DNA glycosylases. Their active sites have a structure that can only bind extrahelical target bases, as observed in the crystal structure of human uracil-DNA glycosylase in a complex with double-stranded DNA. Nucleotide flipping is apparently actively facilitated by the enzyme. With bacteriophage T4 endonuclease V, a pyrimidine-dimer glycosylase, the enzyme gains access to the target base by flipping out an adenine opposite to the dimer. A conserved helix–hairpin–helix motif and an invariant Asp residue are found in the active sites of more than 20 monofunctional and bifunctional DNA glycosylases. In bifunctional DNA glycosylases, the conserved Asp is thought to deprotonate a conserved Lys, forming an amine nucleophile. The nucleophile forms a covalent intermediate (Schiff base) with the deoxyribose anomeric carbon and expels the base. Deoxyribose subsequently undergoes several transformations, resulting in strand cleavage and regeneration of the free enzyme. The catalytic mechanism of monofunctional glycosylases does not involve covalent intermediates. Instead the conserved Asp residue may activate a water molecule which acts as the attacking nucleophile.

scholarly journals Pms2 and uracil-DNA glycosylases act jointly in the mismatch repair pathway to generate Ig gene mutations at A-T base pairs

2017 ◽  
Vol 214 (4) ◽  
pp. 1169-1180 ◽  
Author(s):  
Giulia Girelli Zubani ◽  
Marija Zivojnovic ◽  
Annie De Smet ◽  
Olivier Albagli-Curiel ◽  
François Huetz ◽  
...  
Keyword(s):  
Mismatch Repair ◽  
Somatic Hypermutation ◽  
Cytidine Deaminase ◽  
Gene Mutations ◽  
Time Frame ◽  
Dna Glycosylase ◽  
Immunoglobulin Gene ◽  
Dna Glycosylases ◽  
Base Pairs ◽  
Open Question

During somatic hypermutation (SHM) of immunoglobulin genes, uracils introduced by activation-induced cytidine deaminase are processed by uracil-DNA glycosylase (UNG) and mismatch repair (MMR) pathways to generate mutations at G-C and A-T base pairs, respectively. Paradoxically, the MMR-nicking complex Pms2/Mlh1 is apparently dispensable for A-T mutagenesis. Thus, how detection of U:G mismatches is translated into the single-strand nick required for error-prone synthesis is an open question. One model proposed that UNG could cooperate with MMR by excising a second uracil in the vicinity of the U:G mismatch, but it failed to explain the low impact of UNG inactivation on A-T mutagenesis. In this study, we show that uracils generated in the G1 phase in B cells can generate equal proportions of A-T and G-C mutations, which suggests that UNG and MMR can operate within the same time frame during SHM. Furthermore, we show that Ung−/−Pms2−/− mice display a 50% reduction in mutations at A-T base pairs and that most remaining mutations at A-T bases depend on two additional uracil glycosylases, thymine-DNA glycosylase and SMUG1. These results demonstrate that Pms2/Mlh1 and multiple uracil glycosylases act jointly, each one with a distinct strand bias, to enlarge the immunoglobulin gene mutation spectrum from G-C to A-T bases.

scholarly journals Serine protease dynamics revealed by NMR analysis of the thrombin-thrombomodulin complex

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Riley B. Peacock ◽  
Taylor McGrann ◽  
Marco Tonelli ◽  
Elizabeth A. Komives
Keyword(s):  
Active Site ◽  
Serine Proteases ◽  
Protein C ◽  
Catalytic Mechanism ◽  
Bond Cleavage ◽  
Peptide Bond ◽  
Peptide Bond Cleavage ◽  
Exchange Mass Spectrometry ◽  
Catalytically Active ◽  
Hydrogen Deuterium

AbstractSerine proteases catalyze a multi-step covalent catalytic mechanism of peptide bond cleavage. It has long been assumed that serine proteases including thrombin carry-out catalysis without significant conformational rearrangement of their stable two-β-barrel structure. We present nuclear magnetic resonance (NMR) and hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments on the thrombin-thrombomodulin (TM) complex. Thrombin promotes procoagulative fibrinogen cleavage when fibrinogen engages both the anion binding exosite 1 (ABE1) and the active site. It is thought that TM promotes cleavage of protein C by engaging ABE1 in a similar manner as fibrinogen. Thus, the thrombin-TM complex may represent the catalytically active, ABE1-engaged thrombin. Compared to apo- and active site inhibited-thrombin, we show that thrombin-TM has reduced μs-ms dynamics in the substrate binding (S1) pocket consistent with its known acceleration of protein C binding. Thrombin-TM has increased μs-ms dynamics in a β-strand connecting the TM binding site to the catalytic aspartate. Finally, thrombin-TM had doublet peaks indicative of dynamics that are slow on the NMR timescale in residues along the interface between the two β-barrels. Such dynamics may be responsible for facilitating the N-terminal product release and water molecule entry that are required for hydrolysis of the acyl-enzyme intermediate.

scholarly journals Cutting Edge: The G-U Mismatch Glycosylase Methyl-CpG Binding Domain 4 Is Dispensable for Somatic Hypermutation and Class Switch Recombination

2003 ◽  
Vol 170 (4) ◽  
pp. 1620-1624 ◽  
Author(s):  
Philip D. Bardwell ◽  
Alberto Martin ◽  
Edmund Wong ◽  
Ziqiang Li ◽  
Winfried Edelmann ◽  
...  
Keyword(s):  
Somatic Hypermutation ◽  
Class Switch Recombination ◽  
Binding Domain ◽  
Cutting Edge ◽  
Class Switch ◽  
Domain 4

The effect of sequence context on the activity of cytosine DNA glycosylases

2015 ◽  
Vol 11 (12) ◽  
pp. 3273-3278
Author(s):  
Scott T. Kimber ◽  
Tom Brown ◽  
Keith R. Fox
Keyword(s):  
Dna Glycosylase ◽  
Dna Glycosylases ◽  
Sequence Context ◽  
Uracil Dna Glycosylase

We have examined how sequence context affects the ability of (N204D:L272A) mutants of uracil DNA glycosylase to cleave CX mismatches.

scholarly journals Thymine DNA Glycosylase Can Rapidly Excise 5-Formylcytosine and 5-Carboxylcytosine

2011 ◽  
Vol 286 (41) ◽  
pp. 35334-35338 ◽  
Author(s):  
Atanu Maiti ◽  
Alexander C. Drohat
Keyword(s):  
Embryonic Development ◽  
Catalytic Mechanism ◽  
Excision Repair ◽  
Dna Demethylation ◽  
Oxidation Products ◽  
Dna Glycosylase ◽  
Thymine Dna Glycosylase ◽  
Active Demethylation ◽  
Active Dna Demethylation ◽  
Base Excision

Thymine DNA glycosylase (TDG) excises T from G·T mispairs and is thought to initiate base excision repair (BER) of deaminated 5-methylcytosine (mC). Recent studies show that TDG, including its glycosylase activity, is essential for active DNA demethylation and embryonic development. These and other findings suggest that active demethylation could involve mC deamination by a deaminase, giving a G·T mispair followed by TDG-initiated BER. An alternative proposal is that demethylation could involve iterative oxidation of mC to 5-hydroxymethylcytosine (hmC) and then to 5-formylcytosine (fC) and 5-carboxylcytosine (caC), mediated by a Tet (ten eleven translocation) enzyme, with conversion of caC to C by a putative decarboxylase. Our previous studies suggest that TDG could excise fC and caC from DNA, which could provide another potential demethylation mechanism. We show here that TDG rapidly removes fC, with higher activity than for G·T mispairs, and has substantial caC excision activity, yet it cannot remove hmC. TDG excision of fC and caC, oxidation products of mC, is consistent with its strong specificity for excising bases from a CpG context. Our findings reveal a remarkable new aspect of specificity for TDG, inform its catalytic mechanism, and suggest that TDG could protect against fC-induced mutagenesis. The results also suggest a new potential mechanism for active DNA demethylation, involving TDG excision of Tet-produced fC (or caC) and subsequent BER. Such a mechanism obviates the need for a decarboxylase and is consistent with findings that TDG glycosylase activity is essential for active demethylation and embryonic development, as are mechanisms involving TDG excision of deaminated mC or hmC.

scholarly journals Embryonic extracts derived from the nematode Caenorhabditis elegans remove uracil from DNA by the sequential action of uracil-DNA glycosylase and AP (apurinic/apyrimidinic) endonuclease

2002 ◽  
Vol 365 (2) ◽  
pp. 547-553 ◽  
Author(s):  
Andrea SHATILLA ◽  
Dindial RAMOTAR
Keyword(s):  
Caenorhabditis Elegans ◽  
Excision Repair ◽  
Specific Inhibitor ◽  
Dna Glycosylase ◽  
Dependent Manner ◽  
Nematode Caenorhabditis Elegans ◽  
Uracil Dna Glycosylase ◽  
Ap Endonuclease ◽  
C Elegans ◽  
Ap Site

DNA bases continuously undergo modifications in response to endogenous reactions such as oxidation, alkylation or deamination. The modified bases are primarily removed by DNA glycosylases, which cleave the N-glycosylic bond linking the base to the sugar, to generate an apurinic/apyrimidinic (AP) site, and this latter lesion is highly mutagenic. Previously, no study has demonstrated the processing of these lesions in the nematode Caenorhabditis elegans. Herein, we report the existence of uracil-DNA glycosylase and AP endonuclease activities in extracts derived from embryos of C. elegans. These enzyme activities were monitored using a defined 5′-end 32P-labelled 42-bp synthetic oligonucleotide substrate bearing a single uracil residue opposite guanine at position 21. The embryonic extract rapidly cleaved the substrate in a time-dependent manner to produce a 20-mer product. The extract did not excise adenine or thymine opposite guanine, although uracil opposite either adenine or thymine was processed. Addition of the highly specific inhibitor of uracil-DNA glycosylase produced by Bacillus subtilis to the extract prevented the formation of the 20-mer product, indicating that removal of uracil is catalysed by uracil-DNA glycosylase. The data suggest that the 20-mer product was generated by a sequential reaction, i.e., removal of the uracil base followed by 5′-cleavage of the AP site. Further analysis revealed that product formation was dependent upon the presence of Mg2+, suggesting that cleavage of the AP site, following uracil excision, is carried out by a Mg2+-dependent AP endonuclease. It would appear that these activities correspond to the first two steps of a putative base-excision-repair pathway in C. elegans.

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