Brandt F Eichman | Vanderbilt University (original) (raw)

Papers by Brandt F Eichman

Introduction-Poly(ADP-ribose) polymerase-1 (PARP-1) is an abundant enzyme in the cell nucleus tha... more Introduction-Poly(ADP-ribose) polymerase-1 (PARP-1) is an abundant enzyme in the cell nucleus that regulates genome repair by binding to DNA damage sites and creating the poly(ADPribose) posttranslational modification. PARP-1 hyperactivity leads to cell stress/death associated with many prominent diseases (e.g. cardiovascular disease and several common neurodegenerative disorders). PARP-1 has notably emerged as an effective clinical target for a growing list of

Nature Communications, 2021

Microbes produce a broad spectrum of antibiotic natural products, including many DNA-damaging gen... more Microbes produce a broad spectrum of antibiotic natural products, including many DNA-damaging genotoxins. Among the most potent of these are DNA alkylating agents in the spirocyclopropylcyclohexadienone (SCPCHD) family, which includes the duocarmycins, CC-1065, gilvusmycin, and yatakemycin. The yatakemycin biosynthesis cluster in Streptomyces sp. TP-A0356 contains an AlkD-related DNA glycosylase, YtkR2, that serves as a self-resistance mechanism against yatakemycin toxicity. We previously reported that AlkD, which is not present in an SCPCHD producer, provides only limited resistance against yatakemycin. We now show that YtkR2 and C10R5, a previously uncharacterized homolog found in the CC-1065 biosynthetic gene cluster of Streptomyces zelensis, confer far greater resistance against their respective SCPCHD natural products. We identify a structural basis for substrate specificity across gene clusters and show a correlation between in vivo resistance and in vitro enzymatic activity i...

Nucleic Acids Research, 2020

Interstrand DNA crosslinks (ICLs) are a toxic form of DNA damage that block DNA replication and t... more Interstrand DNA crosslinks (ICLs) are a toxic form of DNA damage that block DNA replication and transcription by tethering the opposing strands of DNA. ICL repair requires unhooking of the tethered strands by either nuclease incision of the DNA backbone or glycosylase cleavage of the crosslinked nucleotide. In bacteria, glycosylase-mediated ICL unhooking was described in Streptomyces as a means of self-resistance to the genotoxic natural product azinomycin B. The mechanistic details and general utility of glycosylase-mediated ICL repair in other bacteria are unknown. Here, we identify the uncharacterized Escherichia coli protein YcaQ as an ICL repair glycosylase that protects cells against the toxicity of crosslinking agents. YcaQ unhooks both sides of symmetric and asymmetric ICLs in vitro, and loss or overexpression of ycaQ sensitizes E. coli to the nitrogen mustard mechlorethamine. Comparison of YcaQ and UvrA-mediated ICL resistance mechanisms establishes base excision as an alternate ICL repair pathway in bacteria.

DNA Repair, 2020

Abasic (AP) sites are one of the most frequently occurring types of DNA damage. They lead to DNA ... more Abasic (AP) sites are one of the most frequently occurring types of DNA damage. They lead to DNA strand breaks, interstrand DNA crosslinks, and block transcription and replication. Mutagenicity of AP sites arises from translesion synthesis (TLS) by error-prone bypass polymerases. Recently, a new cellular response to AP sites was discovered, in which the protein HMCES (5-hydroxymethlycytosine (5hmC) binding, embryonic stem cell-specific) forms a stable, covalent DNA-protein crosslink (DPC) to AP sites at stalled replication forks. The stability of the HMCES-DPC prevents strand cleavage by endonucleases and mutagenic bypass by TLS polymerases. Crosslinking is carried out by a unique SRAP (SOS Response Associated Peptidase) domain conserved across all domains of life. Here, we review the collection of recently reported SRAP crystal structures from human HMCES and E. coli YedK, which provide a unified basis for SRAP specificity and a putative chemical mechanism of AP site crosslinking. We discuss the structural and chemical basis for the stability of the SRAP DPC and how it differs from covalent protein-DNA intermediates in DNA lyase catalysis of strand scission. 1.2. AP site repair and tolerance Despite the fact that AP sites form more readily in single-stranded DNA (ssDNA) [7,20], AP site repair occurs within the context of doublestranded DNA (dsDNA) as part of BER [8,21,22]. In the second step of

Trends in Biochemical Sciences, 2019

Recent studies revealed new and unexpected functions for diverse DNA glycosylases, expanding the ... more Recent studies revealed new and unexpected functions for diverse DNA glycosylases, expanding the known roles of the BER pathway.

BioEssays, 2018

DNA glycosylases remove aberrant DNA nucleobases as the first enzymatic step of the base excision... more DNA glycosylases remove aberrant DNA nucleobases as the first enzymatic step of the base excision repair (BER) pathway. The alkyl-DNA glycosylases AlkC and AlkD adopt a unique structure based on α-helical HEAT repeats. Both enzymes identify and excise their substrates without a base-flipping mechanism used by other glycosylases and nucleic acid processing proteins to access nucleobases that are otherwise stacked inside the double-helix. Consequently, these glycosylases act on a variety of cationic nucleobase modifications, including bulky adducts, not previously associated with BER. The related non-enzymatic HEAT-like repeat (HLR) proteins, AlkD2, and AlkF, have unique nucleic acid binding properties that expand the functions of this relatively new protein superfamily beyond DNA repair. Here, we review the phylogeny, biochemistry, and structures of the HLR proteins, which have helped broaden our understanding of the mechanisms by which DNA glycosylases locate and excise chemically modified DNA nucleobases.

The EMBO journal, Jan 20, 2017

DNA glycosylases preserve genome integrity and define the specificity of the base excision repair... more DNA glycosylases preserve genome integrity and define the specificity of the base excision repair pathway for discreet, detrimental modifications, and thus, the mechanisms by which glycosylases locate DNA damage are of particular interest. Bacterial AlkC and AlkD are specific for cationic alkylated nucleobases and have a distinctive HEAT-like repeat (HLR) fold. AlkD uses a unique non-base-flipping mechanism that enables excision of bulky lesions more commonly associated with nucleotide excision repair. In contrast, AlkC has a much narrower specificity for small lesions, principally N3-methyladenine (3mA). Here, we describe how AlkC selects for and excises 3mA using a non-base-flipping strategy distinct from that of AlkD. A crystal structure resembling a catalytic intermediate complex shows how AlkC uses unique HLR and immunoglobulin-like domains to induce a sharp kink in the DNA, exposing the damaged nucleobase to active site residues that project into the DNA This active site can a...

Proceedings of the National Academy of Sciences, 2017

Significance DNA glycosylases are important repair enzymes that safeguard the integrity of the ge... more Significance DNA glycosylases are important repair enzymes that safeguard the integrity of the genome by excising chemically damaged DNA bases from the phosphoribose backbone. Recently, these enzymes were found to repair DNA interstrand cross-links (ICLs). ICLs are highly toxic DNA lesions formed by various bifunctional metabolites, environmental toxins, and chemotherapeutic agents that block normal DNA metabolism. This work describes the crystal structure of a newly discovered bacterial DNA glycosylase that repairs ICLs formed by azinomycin B, a potent antimicrobial and antitumor agent. The protein belongs to a structural superfamily prevalent in pathogenic bacteria and may serve as an important therapeutic target.

Nature Chemical Biology, 2017

Yatakemycin (YTM) is an extraordinarily toxic DNA alkylating agent with potent antimicrobial and ... more Yatakemycin (YTM) is an extraordinarily toxic DNA alkylating agent with potent antimicrobial and antitumor properties and the most recent addition to the CC-1065 and duocarmycin family of natural products. While bulky DNA lesions the size of those produced by YTM are normally removed from the genome by the nucleotide excision repair (NER) pathway, YTM adducts are also a substrate for the bacterial DNA glycosylases AlkD and YtkR2, unexpectedly implicating base excision repair (BER) in their elimination. The reason for the extreme toxicity of these lesions and the molecular basis for how they are eliminated by BER have been unclear. Here, we describe the structural and biochemical properties of YTM adducts responsible for their toxicity, and define the mechanism by which they are excised by AlkD. These findings delineate an alternative strategy for repair of bulky DNA damage and establish the cellular utility of this pathway relative to that of NER.

Parlare dell'ultimo decennio e del rapporto con Weimar e Bonn significa ripercorrere i dati della... more Parlare dell'ultimo decennio e del rapporto con Weimar e Bonn significa ripercorrere i dati della grande recessione post-2009, che ha colpito tutto il mondo occidentale ma soprattutto l'Europa di Lisbona, e analizzare l'evolversi della crisi italiana. Benchè le opere su Weimar non siano state numerose, la sensazione di un avvitarsi weimariano della situazione europea ed italiana è sempre più presente.

Nature, Jan 12, 2015

Threats to genomic integrity arising from DNA damage are mitigated by DNA glycosylases, which ini... more Threats to genomic integrity arising from DNA damage are mitigated by DNA glycosylases, which initiate the base excision repair pathway by locating and excising aberrant nucleobases. How these enzymes find small modifications within the genome is a current area of intensive research. A hallmark of these and other DNA repair enzymes is their use of base flipping to sequester modified nucleotides from the DNA helix and into an active site pocket. Consequently, base flipping is generally regarded as an essential aspect of lesion recognition and a necessary precursor to base excision. Here we present the first, to our knowledge, DNA glycosylase mechanism that does not require base flipping for either binding or catalysis. Using the DNA glycosylase AlkD from Bacillus cereus, we crystallographically monitored excision of an alkylpurine substrate as a function of time, and reconstructed the steps along the reaction coordinate through structures representing substrate, intermediate and prod...

PLOS ONE, 2015

DNA glycosylases are important repair enzymes that eliminate a diverse array of aberrant nucleoba... more DNA glycosylases are important repair enzymes that eliminate a diverse array of aberrant nucleobases from the genomes of all organisms. Individual bacterial species often contain multiple paralogs of a particular glycosylase, yet the molecular and functional distinctions between these paralogs are not well understood. The recently discovered HEAT-like repeat (HLR) DNA glycosylases are distributed across all domains of life and are distinct in their specificity for cationic alkylpurines and mechanism of damage recognition. Here, we describe a number of phylogenetically diverse bacterial species with two orthologs of the HLR DNA glycosylase AlkD. One ortholog, which we designate AlkD2, is substantially less conserved. The crystal structure of Streptococcus mutans AlkD2 is remarkably similar to AlkD but lacks the only helix present in AlkD that penetrates the DNA minor groove. We show that AlkD2 possesses only weak DNA binding affinity and lacks alkylpurine excision activity. Mutational analysis of residues along this DNA binding helix in AlkD substantially reduced binding affinity for damaged DNA, for the first time revealing the importance of this structural motif for damage recognition by HLR glycosylases.

Biochemistry, Jan 10, 2015

This is an open access article published under an ACS AuthorChoice License, which permits copying... more This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.

Biochemistry, Jan 22, 2014

Methylation of cytosine to 5-methylcytosine (5mC) is important for gene expression, gene imprinti... more Methylation of cytosine to 5-methylcytosine (5mC) is important for gene expression, gene imprinting, X-chromosome inactivation, and transposon silencing. Active demethylation in animals is believed to proceed by DNA glycosylase removal of deaminated or oxidized 5mC. In plants, 5mC is removed from the genome directly by the DEMETER (DME) family of DNA glycosylases. Arabidopsis thaliana DME excises 5mC to activate expression of maternally imprinted genes. Although the related Repressor of Silencing 1 (ROS1) enzyme has been characterized, the molecular basis for 5mC recognition by DME has not been investigated. Here, we present a structure-function analysis of DME and the related DME-like 3 (DML3) glycosylases for 5mC and its oxidized derivatives. Relative to 5mC, DME and DML3 exhibited robust activity toward 5-hydroxymethylcytosine, limited activity for 5-carboxylcytosine, and no activity for 5-formylcytosine. We used homology modeling and mutational analysis of base excision and DNA ...

Biochemistry, 2015

5-Hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) form during... more 5-Hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) form during active demethylation of 5methylcytosine (5mC) and are implicated in epigenetic regulation of the genome. They are differentially processed by thymine DNA glycosylase (TDG), an enzyme involved in active demethylation of 5mC. Three modified Dickerson−Drew dodecamer (DDD) sequences, amenable to crystallographic and spectroscopic analyses and containing the 5′-CG-3′ sequence associated with genomic cytosine methylation, containing 5hmC, 5fC, or 5caC placed site-specifically into the 5′-T 8 X 9 G 10-3′ sequence of the DDD, were compared. The presence of 5caC at the X 9 base increased the stability of the DDD, whereas 5hmC or 5fC did not. Both 5hmC and 5fC increased imino proton exchange rates and calculated rate constants for base pair opening at the neighboring base pair A 5 :T 8 , whereas 5caC did not. At the oxidized base pair G 4 :X 9 , 5fC exhibited an increase in the imino proton exchange rate and the calculated k op. In all cases, minimal effects to imino proton exchange rates occurred at the neighboring base pair C 3 :G 10. No evidence was observed for imino tautomerization, accompanied by wobble base pairing, for 5hmC, 5fC, or 5caC when positioned at base pair G 4 :X 9 ; each favored Watson−Crick base pairing. However, both 5fC and 5caC exhibited intranucleobase hydrogen bonding between their formyl or carboxyl oxygens, respectively, and the adjacent cytosine N 4 exocyclic amines. The lesion-specific differences observed in the DDD may be implicated in recognition of 5hmC, 5fC, or 5caC in DNA by TDG. However, they do not correlate with differential excision of 5hmC, 5fC, or 5caC by TDG, which may be mediated by differences in transition states of the enzyme-bound complexes.

Cytosine methylation or bromination of the DNA sequence d(GGCGCC) 2 is shown here to induce a nov... more Cytosine methylation or bromination of the DNA sequence d(GGCGCC) 2 is shown here to induce a novel extended and eccentric double helix, which we call E-DNA. Like B-DNA, E-DNA has a long helical rise and bases perpendicular to the helix axis. However, the 3′-endo sugar conformation gives the characteristic deep major groove and shallow minor groove of A-DNA. Also, if allowed to crystallize for a period of time longer than that yielding E-DNA, the methylated sequence forms standard A-DNA, suggesting that E-DNA is a kinetically trapped intermediate in the transition to A-DNA. Thus, the structures presented here chart a crystallographic pathway from B-DNA to A-DNA through the E-DNA intermediate in a single sequence. The E-DNA surface is highly accessible to solvent, with waters in the major groove sitting on exposed faces of the stacked nucleotides. We suggest that the geometry of the waters and the stacked base pairs would promote the spontaneous deamination of 5-methylcytosine in the transition mutation of dm 5 C-dG to dT-dA base pairs. 5-Methyldeoxycytidine (dm 5 C) is often considered the fifth nucleotide of the genetic code. Prokaryotes use cytosine methy-Oregon American Cancer Society, and the Environmental Heath Science Center at OSU. X-ray facilities were funded in part by the M.J. Murdock Charitable Trust.

The EMBO Journal, 2007

DNA glycosylases help maintain the genome by excising chemically modified bases from DNA. Escheri... more DNA glycosylases help maintain the genome by excising chemically modified bases from DNA. Escherichia coli 3methyladenine DNA glycosylase I (TAG) specifically catalyzes the removal of the cytotoxic lesion 3-methyladenine (3mA). The molecular basis for the enzymatic recognition and removal of 3mA from DNA is currently a matter of speculation, in part owing to the lack of a structure of a 3mA-specific glycosylase bound to damaged DNA. Here, high-resolution crystal structures of Salmonella typhi TAG in the unliganded form and in a ternary product complex with abasic DNA and 3mA nucleobase are presented. Despite its structural similarity to the helix-hairpinhelix superfamily of DNA glycosylases, TAG has evolved a modified strategy for engaging damaged DNA. In contrast to other glycosylase-DNA structures, the abasic ribose is not flipped into the TAG active site. This is the first structural demonstration that conformational relaxation must occur in the DNA upon base hydrolysis. Together with mutational studies of TAG enzymatic activity, these data provide a model for the specific recognition and hydrolysis of 3mA from DNA.

Proceedings of the National Academy of Sciences, 2014

Significance Mutations in SMARCAL1, a DNA remodeling protein fundamental to genome integrity duri... more Significance Mutations in SMARCAL1, a DNA remodeling protein fundamental to genome integrity during replication, lead to the developmental disorder Schimke immuno-osseous dysplasia (SIOD). SMARCAL1-deficient cells exhibit collapsed replication forks, S-phase cell cycle arrest, increased chromosomal breaks, hypersensitivity to genotoxic agents, and chromosomal instability. SMARCAL1 facilitates replication restart by catalyzing ATP-dependent fork regression and branch migration, the mechanism of which is poorly understood. Here we provide structural and biochemical evidence for a conserved substrate recognition domain in DNA repair enzymes that couples ATP hydrolysis to remodeling of a variety of DNA structures, and provide insight into the role of this domain in replication fork stability and genome integrity.

Proceedings of the National Academy of Sciences, 2000

Holliday junctions are important structural intermediates in recombination, viral integration, an... more Holliday junctions are important structural intermediates in recombination, viral integration, and DNA repair. We present here the single-crystal structure of the inverted repeat sequence d(CCGGTACCGG) as a Holliday junction at the nominal resolution of 2.1 Å. Unlike the previous crystal structures, this DNA junction has B-DNA arms with all standard Watson–Crick base pairs; it therefore represents the intermediate proposed by Holliday as being involved in homologous recombination. The junction is in the stacked-X conformation, with two interconnected duplexes formed by coaxially stacked arms, and is crossed at an angle of 41.4° as a right-handed X. A sequence comparison with previous B-DNA and junction crystal structures shows that an ACC trinucleotide forms the core of a stable junction in this system. The 3′-C⋅G base pair of this ACC core forms direct and water-mediated hydrogen bonds to the phosphates at the crossover strands. Interactions within this core define the conformation...