Ashok S. Bhagwat

Phylogenomic identification of five new human homologs of the DNA repair enzyme AlkB

BackgroundCombination of biochemical and bioinformatic analyses led to the discovery of oxidative demethylation – a novel DNA repair mechanism catalyzed by the Escherichia coli AlkB protein and its two human homologs, hABH2 and hABH3. This discovery was based on the prediction made by Aravind and Koonin that AlkB is a member of the 2OG-Fe2+ oxygenase superfamily.ResultsIn this article, we report identification and sequence analysis of five human members of the (2OG-Fe2+) oxygenase superfamily designated here as hABH4 through hABH8. These experimentally uncharacterized and poorly annotated genes were not associated with the AlkB family in any database, but are predicted here to be phylogenetically and functionally related to the AlkB family (and specifically to the lineage that groups together hABH2 and hABH3) rather than to any other oxygenase family. Our analysis reveals the history of ABH gene duplications in the evolution of vertebrate genomes.ConclusionsWe hypothesize that hABH 4–8 could either be back-up enzymes for hABH1-3 or may code for novel DNA or RNA repair activities. For example, enzymes that can dealkylate N3-methylpurines or N7-methylpurines in DNA have not been described. Our analysis will guide experimental confirmation of these novel human putative DNA repair enzymes.

The COMBREX Project: Design, Methodology, and Initial Results

Experimental data exists for only a vanishingly small fraction of sequenced microbial genes. This community page discusses the progress made by the COMBREX project to address this important issue using both computational and experimental resources.

Excision of 5-hydroxymethyluracil and 5-carboxylcytosine by the thymine DNA glycosylase domain: its structural basis and implications for active DNA demethylation

The mammalian thymine DNA glycosylase (TDG) is implicated in active DNA demethylation via the base excision repair pathway. TDG excises the mismatched base from G:X mismatches, where X is uracil, thymine or 5-hydroxymethyluracil (5hmU). These are, respectively, the deamination products of cytosine, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). In addition, TDG excises the Tet protein products 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) but not 5hmC and 5mC, when paired with a guanine. Here we present a post-reactive complex structure of the human TDG domain with a 28-base pair DNA containing a G:5hmU mismatch. TDG flips the target nucleotide from the double-stranded DNA, cleaves the N-glycosidic bond and leaves the C1′ hydrolyzed abasic sugar in the flipped state. The cleaved 5hmU base remains in a binding pocket of the enzyme. TDG allows hydrogen-bonding interactions to both T/U-based (5hmU) and C-based (5caC) modifications, thus enabling its activity on a wider range of substrates. We further show that the TDG catalytic domain has higher activity for 5caC at a lower pH (5.5) as compared to the activities at higher pH (7.5 and 8.0) and that the structurally related Escherichia coli mismatch uracil glycosylase can excise 5caC as well. We discuss several possible mechanisms, including the amino-imino tautomerization of the substrate base that may explain how TDG discriminates against 5hmC and 5mC.

Very short patch repair: reducing the cost of cytosine methylation

In Escherichia coli and related bacteria, the product of gene dcm methylates the second cytosine of 5′‐CCWGG sequences (where W is A or T). Deamination of 5‐methylcytosine (5meC) results in C to T mutations. The mutagenic potential of 5meC is reduced by a system called very short patch (VSP) repair, which replaces T with C. T:G and U:G mispairs in the methylatable sequence and in related sequences are recognized by the product of vsr, a gene adjacent to dcm. Vsr creates a nick just 5′ of the mispaired pyrimidine to initiate the repair. Additional products known to be required for VSP repair are DNA polymerase I and DNA ligase. MutS and MutL have a stimulatory role but are not required. The ability of Vsr to recognize T:G mispairs in sequences related to CCWGG is probably responsible for over‐ and under‐representation of certain tetranucleotides in the E. coli genome. Although VSP repair reduces spontaneous mutations at 5meCs in replicating bacteria, mutation hot‐spots persist at these sites. Under conditions that more accurately mimic the natural environment of E. coli, VSP repair appears to be effective in preventing mutation at 5meC.

Efficient deamination of 5-methylcytosines in DNA by human APOBEC3A, but not by AID or APOBEC3G

The AID/APOBEC family of enzymes in higher vertebrates converts cytosines in DNA or RNA to uracil. They play a role in antibody maturation and innate immunity against viruses, and have also been implicated in the demethylation of DNA during early embryogenesis. This is based in part on reported ability of activation-induced deaminase (AID) to deaminate 5-methylcytosines (5mC) to thymine. We have reexamined this possibility for AID and two members of human APOBEC3 family using a novel genetic system in Escherichia coli. Our results show that while all three genes show strong ability to convert C to U, only APOBEC3A is an efficient deaminator of 5mC. To confirm this, APOBEC3A was purified partially and used in an in vitro deamination assay. We found that APOBEC3A can deaminate 5mC efficiently and this activity is comparable to its C to U deamination activity. When the DNA-binding segment of AID was replaced with the corresponding segment from APOBEC3A, the resulting hybrid had much higher ability to convert 5mC to T in the genetic assay. These and other results suggest that the human AID deaminates 5mCs only weakly because the 5-methyl group fits poorly in its DNA-binding pocket.

Mismatch Repair in Methylated DNA STRUCTURE AND ACTIVITY OF THE MISMATCH-SPECIFIC THYMINE GLYCOSYLASE DOMAIN OF METHYL-CpG-BINDING PROTEIN MBD4

MBD4 is a member of the methyl-CpG-binding protein family. It contains two DNA binding domains, an amino-proximal methyl-CpG binding domain (MBD) and a C-terminal mismatch-specific glycosylase domain. Limited in vitro proteolysis of mouse MBD4 yields two stable fragments: a 139-residue fragment including the MBD, and the other 155-residue fragment including the glycosylase domain. Here we show that the latter fragment is active as a glycosylase on a DNA duplex containing a G:T mismatch within a CpG sequence context. The crystal structure confirmed the C-terminal domain is a member of the helix-hairpin-helix DNA glycosylase superfamily. The MBD4 active site is situated in a cleft that likely orients and binds DNA. Modeling studies suggest the mismatched target nucleotide will be flipped out into the active site where candidate residues for catalysis and substrate specificity are present.

Principal causes of hot spots for cytosine to thymine mutations at sites of cytosine methylation in growing cells. A model, its experimental support and implications.

In Escherichia coli and human cells, many sites of cytosine methylation in DNA are hot spots for C to T mutations. It is generally believed that T.G mismatches created by the hydrolytic deamination of 5-methylcytosines (5meC) are intermediates in the mutagenic pathway. A number of hypotheses have been proposed regarding the source of the mispaired thymine and how the cells deal with the mispairs. We have constructed a genetic reversion assay that utilizes a gene on a mini-F to compare the frequency of occurrence of C to T mutations in different genetic backgrounds in exponentially growing E. coli. The results identify at least two causes for the hot spot at a 5meC: (1) the higher rate of deamination of 5meC compared to C generates more T.G than uracil.G (U.G) mismatches, and (2) inefficient repair of T.G mismatches by the very short-patch (VSP) repair system compared to the repair of U. G mismatches by the uracil-DNA glycosylase (Ung). This combination of increased DNA damage when the cytosines are methylated coupled with the relative inefficiency in the post-replicative repair of T.G mismatches can be quantitatively modeled to explain the occurrence of the hot spot at 5meC. This model has implications for mutational hot and cold spots in all organisms.

Determinants of Sequence-Specificity within Human AID and APOBEC3G

Human APOBEC3G (A3G) and activation-induced deaminase (AID) belong to a family of DNA-cytosine deaminases. While A3G targets the last C in a run of Cs, AID targets C in the consensus sequence WRC (W is A or T and R is a purine). Guided by the structures of the A3G carboxyl-terminal catalytic domain (A3G-CTD), we identified two potential regions (region 1 and region 2) that may interact with DNA and swapped the corresponding regions between a variant of A3G-CTD and AID. The resulting hybrids were expressed in Escherichia coli and two different genetic assays and a biochemical assay were used to determine the sequence selectivity of the hybrids in promoting C to T mutations. The results show that while the 10 amino acid region 2 of A3G was its principal sequence-specificity determinant, region 1 of A3G enhanced the target cytosine preference conferred by region 2. In contrast, neither of the two regions in AID individually or in combination were sufficient to confer the DNA sequence preference of this protein upon A3G. Instead, introduction of AID sequences in A3G relaxed the sequence-specificity of the latter protein. Our results show that the sequence selectivity of APOBEC family of enzymes is determined by at least two separate sequence segments and there may be additional regions of the protein involved in DNA sequence recognition.

Cooperation and competition in mismatch repair: Very short-patch repair and methyl-directed mismatch repair in Escherichia coli

In Escherichia coli and related enteric bacteria, repair of base‐base mismatches is performed by two overlapping biochemical processes, methyl‐directed mismatch repair (MMR) and very short‐patch (VSP) repair. While MMR repairs replication errors, VSP repair corrects to C•G mispairs created by 5‐methylcytosine deamination to T. The efficiency of the two pathways changes during the bacterial life cycle; MMR is more efficient during exponential growth and VSP repair is more efficient during the stationary phase. VSP repair and MMR share two proteins, MutS and MutL, and although the two repair pathways are not equally dependent on these proteins, their dual use creates a competition within the cells between the repair processes. The structural and biochemical data on the endonuclease that initiates VSP repair, Vsr, suggest that this protein plays a role similar to MutH (also an endonuclease) in MMR. Biochemical and genetic studies of the two repair pathways have helped eliminate certain models for MMR and put restrictions on models that can be developed regarding either repair process. We review here recent information about the biochemistry of both repair processes and describe the balancing act performed by cells to optimize the competing processes during different phases of the bacterial life cycle.

The role of the Escherichia coli mug protein in the removal of uracil and 3,N(4)-ethenocytosine from DNA.

The human thymine-DNA glycosylase has a sequence homolog in Escherichia coli that is described to excise uracils from U·G mismatches (Gallinari, P., and Jiricny, J. (1996)Nature 383, 735–738) and is named mismatched uracil glycosylase (Mug). It has also been described to remove 3,N 4-ethenocytosine (εC) from εC·G mismatches (Saparbaev, M., and Laval, J. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8508–8513). We used a mug mutant to clarify the role of this protein in DNA repair and mutation avoidance. We find that inactivation of mug has no effect on C to T or 5-methylcytosine to T mutations in E. coli and that this contrasts with the effect of ung defect on C to T mutations and of vsr defect on 5-methylcytosine to T mutations. Even under conditions where it is overproduced in cells, Mug has little effect on the frequency of C to T mutations. Because uracil-DNA glycosylase (Ung) and Vsr are known to repair U·G and T·G mismatches, respectively, we conclude that Mug does not repair U·G or T·G mismatches in vivo. A defect inmug also has little effect on forward mutations, suggesting that Mug does not play a role in avoiding mutations due to endogenous damage to DNA in growing E. coli. Cell-free extracts frommug + ung cells show very little ability to remove uracil from DNA, but can excise εC. The latter activity is missing in extracts from mug cells, suggesting that Mug may be the only enzyme in E. coli that can remove this mutagenic adduct. Thus, the principal role of Mug in E. coli may be to help repair damage to DNA caused by exogenous chemical agents such as chloroacetaldehyde.