RESUMEN
Abasic sites are one of the most common DNA lesions. All known abasic site repair mechanisms operate only when the damage is in double-stranded DNA. Here, we report the discovery of 5-hydroxymethylcytosine (5hmC) binding, ESC-specific (HMCES) as a sensor of abasic sites in single-stranded DNA. HMCES acts at replication forks, binds PCNA and single-stranded DNA, and generates a DNA-protein crosslink to shield abasic sites from error-prone processing. This unusual HMCES DNA-protein crosslink intermediate is resolved by proteasome-mediated degradation. Acting as a suicide enzyme, HMCES prevents translesion DNA synthesis and the action of endonucleases that would otherwise generate mutations and double-strand breaks. HMCES is evolutionarily conserved in all domains of life, and its biochemical properties are shared with its E. coli ortholog. Thus, HMCES is an ancient DNA lesion recognition protein that preserves genome integrity by promoting error-free repair of abasic sites in single-stranded DNA.
Asunto(s)
5-Metilcitosina/análogos & derivados , Reparación del ADN/fisiología , ADN de Cadena Simple/fisiología , 5-Metilcitosina/metabolismo , Ácido Apurínico/metabolismo , ADN/metabolismo , Daño del ADN/fisiología , Replicación del ADN/fisiología , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endonucleasas , Escherichia coli/metabolismo , Polinucleótidos/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismoRESUMEN
G-quadruplexes (G4s) form throughout the genome and influence important cellular processes. Their deregulation can challenge DNA replication fork progression and threaten genome stability. Here, we demonstrate an unexpected role for the double-stranded DNA (dsDNA) translocase helicase-like transcription factor (HLTF) in responding to G4s. We show that HLTF, which is enriched at G4s in the human genome, can directly unfold G4s in vitro and uses this ATP-dependent translocase function to suppress G4 accumulation throughout the cell cycle. Additionally, MSH2 (a component of MutS heterodimers that bind G4s) and HLTF act synergistically to suppress G4 accumulation, restrict alternative lengthening of telomeres, and promote resistance to G4-stabilizing drugs. In a discrete but complementary role, HLTF restrains DNA synthesis when G4s are stabilized by suppressing primase-polymerase (PrimPol)-dependent repriming. Together, the distinct roles of HLTF in the G4 response prevent DNA damage and potentially mutagenic replication to safeguard genome stability.
RESUMEN
DNA replication stress can stall replication forks, leading to genome instability. DNA damage tolerance pathways assist fork progression, promoting replication fork reversal, translesion DNA synthesis (TLS), and repriming. In the absence of the fork remodeler HLTF, forks fail to slow following replication stress, but underlying mechanisms and cellular consequences remain elusive. Here, we demonstrate that HLTF-deficient cells fail to undergo fork reversal in vivo and rely on the primase-polymerase PRIMPOL for repriming, unrestrained replication, and S phase progression upon limiting nucleotide levels. By contrast, in an HLTF-HIRAN mutant, unrestrained replication relies on the TLS protein REV1. Importantly, HLTF-deficient cells also exhibit reduced double-strand break (DSB) formation and increased survival upon replication stress. Our findings suggest that HLTF promotes fork remodeling, preventing other mechanisms of replication stress tolerance in cancer cells. This remarkable plasticity of the replication fork may determine the outcome of replication stress in terms of genome integrity, tumorigenesis, and response to chemotherapy.
Asunto(s)
Replicación del ADN/fisiología , Proteínas de Unión al ADN/metabolismo , ADN/biosíntesis , Factores de Transcripción/metabolismo , Línea Celular Tumoral , ADN/genética , Daño del ADN/genética , ADN Primasa/metabolismo , ADN Primasa/fisiología , Reparación del ADN/genética , Replicación del ADN/genética , Proteínas de Unión al ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , ADN Polimerasa Dirigida por ADN/fisiología , Células HEK293 , Humanos , Células K562 , Enzimas Multifuncionales/metabolismo , Enzimas Multifuncionales/fisiología , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/fisiología , Factores de Transcripción/genéticaRESUMEN
Maintenance of DNA integrity is essential to all forms of life. DNA damage generated by reaction with genotoxic chemicals results in deleterious mutations, genome instability, and cell death. Pathogenic bacteria encounter several genotoxic agents during infection. In keeping with this, the loss of DNA repair networks results in virulence attenuation in several bacterial species. Interstrand DNA crosslinks (ICLs) are a type of DNA lesion formed by covalent linkage of opposing DNA strands and are particularly toxic as they interfere with replication and transcription. Bacteria have evolved specialized DNA glycosylases that unhook ICLs, thereby initiating their repair. In this study, we describe AlkX, a DNA glycosylase encoded by the multidrug resistant pathogen Acinetobacter baumannii. AlkX exhibits ICL unhooking activity similar to that of its Escherichia coli homolog YcaQ. Interrogation of the in vivo role of AlkX revealed that its loss sensitizes cells to DNA crosslinking and impairs A. baumannii colonization of the lungs and dissemination to distal tissues during pneumonia. These results suggest that AlkX participates in A. baumannii pathogenesis and protects the bacterium from stress conditions encountered in vivo. Consistent with this, we found that acidic pH, an environment encountered during host colonization, results in A. baumannii DNA damage and that alkX is induced by, and contributes to, defense against acidic conditions. Collectively, these studies reveal functions for a recently described class of proteins encoded in a broad range of pathogenic bacterial species.
Asunto(s)
Acinetobacter baumannii , Daño del ADN , ADN Glicosilasas , Acinetobacter baumannii/patogenicidad , Acinetobacter baumannii/genética , Acinetobacter baumannii/enzimología , Acinetobacter baumannii/metabolismo , ADN Glicosilasas/metabolismo , ADN Glicosilasas/genética , Reparación del ADN , Infecciones por Acinetobacter/microbiología , Infecciones por Acinetobacter/patología , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Animales , Ratones , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Virulencia , Escherichia coli/genética , Escherichia coli/metabolismoRESUMEN
TatD enzymes are evolutionarily conserved deoxyribonucleases associated with DNA repair, apoptosis, development, and parasite virulence. Three TatD paralogs exist in humans, but their nuclease functions are unknown. Here, we describe the nuclease activities of two of the three human TatD paralogs, TATDN1 and TATDN3, which represent two phylogenetically distinct clades based on unique active site motifs. We found that in addition to 3'-5' exonuclease activity associated with other TatD proteins, both TATDN1 and TATDN3 exhibited apurinic/apyrimidinic (AP) endonuclease activity. The AP endonuclease activity was observed only in double-stranded DNA, whereas exonuclease activity was operative primarily in single-stranded DNA. Both nuclease activities were observed in the presence of Mg2+ or Mn2+, and we identified several divalent metal cofactors that inhibited exonuclease and supported AP endonuclease activity. Biochemical analysis and a crystal structure of TATDN1 bound to 2'-deoxyadenosine 5'-monophosphate in the active site are consistent with two-metal ion catalysis, and we identify several residues that differentiate nuclease activities in the two proteins. In addition, we show that the three Escherichia coli TatD paralogs are also AP endonucleases, indicating that this activity is conserved across evolution. Together, these results indicate that TatD enzymes constitute a family of ancient AP endonucleases.
Asunto(s)
Reparación del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Exonucleasas , Humanos , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Endonucleasas/metabolismo , Escherichia coli/metabolismo , Exonucleasas/metabolismoRESUMEN
Two families of DNA glycosylases (YtkR2/AlkD, AlkZ/YcaQ) have been found to remove bulky and crosslinking DNA adducts produced by bacterial natural products. Whether DNA glycosylases eliminate other types of damage formed by structurally diverse antibiotics is unknown. Here, we identify four DNA glycosylases-TxnU2, TxnU4, LldU1 and LldU5-important for biosynthesis of the aromatic polyketide antibiotics trioxacarcin A (TXNA) and LL-D49194 (LLD), and show that the enzymes provide self-resistance to the producing strains by excising the intercalated guanine adducts of TXNA and LLD. These enzymes are highly specific for TXNA/LLD-DNA lesions and have no activity toward other, less stable alkylguanines as previously described for YtkR2/AlkD and AlkZ/YcaQ. Similarly, TXNA-DNA adducts are not excised by other alkylpurine DNA glycosylases. TxnU4 and LldU1 possess unique active site motifs that provide an explanation for their tight substrate specificity. Moreover, we show that abasic (AP) sites generated from TxnU4 excision of intercalated TXNA-DNA adducts are incised by AP endonuclease less efficiently than those formed by 7mG excision. This work characterizes a distinct class of DNA glycosylase acting on intercalated DNA adducts and furthers our understanding of specific DNA repair self-resistance activities within antibiotic producers of structurally diverse, highly functionalized DNA damaging agents.
Asunto(s)
Aductos de ADN , ADN Glicosilasas , Aminoglicósidos , Antibacterianos/farmacología , Daño del ADN , ADN Glicosilasas/metabolismo , Reparación del ADNRESUMEN
The base excision repair (BER) pathway historically has been associated with maintaining genome integrity by eliminating nucleobases with small chemical modifications. In the past several years, however, BER was found to play additional roles in genome maintenance and metabolism, including sequence-specific restriction modification and repair of bulky adducts and interstrand crosslinks. Central to this expanded biological utility are specialized DNA glycosylases - enzymes that selectively excise damaged, modified, or mismatched nucleobases. In this review we discuss the newly identified roles of the BER pathway and examine the structural and mechanistic features of the DNA glycosylases that enable these functions.
Asunto(s)
ADN Glicosilasas/metabolismo , Reparación del ADN , ADN/metabolismo , ADN/química , Daño del ADN , ADN Glicosilasas/química , HumanosRESUMEN
Apurinic/apyrimidinic (AP, or abasic) sites in DNA are one of the most common forms of DNA damage. AP sites are reactive and form cross-links to both proteins and DNA, are prone to strand breakage, and inhibit DNA replication and transcription. The replication-associated AP site repair protein HMCES protects cells from strand breaks, inhibits mutagenic translesion synthesis, and participates in repair of interstrand DNA cross-links derived from AP sites by forming a stable thiazolidine DNA-protein cross-link (DPC) to AP sites in single-stranded DNA (ssDNA). Despite the importance of HMCES to genome maintenance and the evolutionary conservation of its catalytic SRAP (SOS Response Associated Peptidase) domain, the enzymatic mechanisms of DPC formation and resolution are unknown. Using the bacterial homolog YedK, we show that the SRAP domain catalyzes conversion of the AP site to its reactive, ring-opened aldehyde form, and we provide structural evidence for the Schiff base intermediate that forms prior to the more stable thiazolidine. We also report two new activities, whereby SRAP reacts with polyunsaturated aldehydes at DNA 3'-ends generated by bifunctional DNA glycosylases and catalyzes direct reversal of the DPC to regenerate the AP site, the latter of which we observe in both YedK and HMCES-SRAP proteins. Taken together, this work provides insights into possible mechanisms by which HMCES DPCs are resolved in cells.
Asunto(s)
ADN Glicosilasas , ADN de Cadena Simple , Aldehídos , ADN/metabolismo , Daño del ADN , ADN Glicosilasas/metabolismo , Reparación del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Péptido Hidrolasas/metabolismo , Proteínas/genética , Respuesta SOS en Genética , Bases de Schiff , TiazolidinasRESUMEN
Stalled replication forks are a critical problem for the cell because they can lead to complex genome rearrangements that underlie cell death and disease. Processes such as DNA damage tolerance and replication fork reversal protect stalled forks from these events. A central mediator of these DNA damage responses in humans is the Rad5-related DNA translocase, HLTF. Here, we present biochemical and structural evidence that the HIRAN domain, an ancient and conserved domain found in HLTF and other DNA processing proteins, is a modified oligonucleotide/oligosaccharide (OB) fold that binds to 3' ssDNA ends. We demonstrate that the HIRAN domain promotes HLTF-dependent fork reversal in vitro through its interaction with 3' ssDNA ends found at forks. Finally, we show that HLTF restrains replication fork progression in cells in a HIRAN-dependent manner. These findings establish a mechanism of HLTF-mediated fork reversal and provide insight into the requirement for distinct fork remodeling activities in the cell.
Asunto(s)
Replicación del ADN , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión/genética , Western Blotting , Línea Celular Tumoral , Cristalografía por Rayos X , ADN/química , ADN/genética , ADN de Cadena Simple/química , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Humanos , Espectroscopía de Resonancia Magnética , Modelos Genéticos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Conformación de Ácido Nucleico , Unión Proteica , Estructura Terciaria de Proteína , Interferencia de ARN , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
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 accommodate and excise N3-methylcytosine (3mC) and N1-methyladenine (1mA), which are also repaired by AlkB-catalyzed oxidative demethylation, providing a potential alternative mechanism for repair of these lesions in bacteria.
Asunto(s)
Bacillus cereus/enzimología , Aductos de ADN/química , Aductos de ADN/metabolismo , Daño del ADN , ADN Glicosilasas/química , ADN Glicosilasas/metabolismo , Reparación del ADN , Adenina/análogos & derivados , Adenina/química , Alquilación , Secuencia de Aminoácidos , Dominio Catalítico , Cristalografía por Rayos X , Modelos Moleculares , Conformación Proteica , Homología de SecuenciaRESUMEN
Cell-cycle checkpoints and DNA repair processes protect organisms from potentially lethal mutational damage. Compared to other budding yeasts in the subphylum Saccharomycotina, we noticed that a lineage in the genus Hanseniaspora exhibited very high evolutionary rates, low Guanine-Cytosine (GC) content, small genome sizes, and lower gene numbers. To better understand Hanseniaspora evolution, we analyzed 25 genomes, including 11 newly sequenced, representing 18/21 known species in the genus. Our phylogenomic analyses identify two Hanseniaspora lineages, a faster-evolving lineage (FEL), which began diversifying approximately 87 million years ago (mya), and a slower-evolving lineage (SEL), which began diversifying approximately 54 mya. Remarkably, both lineages lost genes associated with the cell cycle and genome integrity, but these losses were greater in the FEL. E.g., all species lost the cell-cycle regulator WHIskey 5 (WHI5), and the FEL lost components of the spindle checkpoint pathway (e.g., Mitotic Arrest-Deficient 1 [MAD1], Mitotic Arrest-Deficient 2 [MAD2]) and DNA-damage-checkpoint pathway (e.g., Mitosis Entry Checkpoint 3 [MEC3], RADiation sensitive 9 [RAD9]). Similarly, both lineages lost genes involved in DNA repair pathways, including the DNA glycosylase gene 3-MethylAdenine DNA Glycosylase 1 (MAG1), which is part of the base-excision repair pathway, and the DNA photolyase gene PHotoreactivation Repair deficient 1 (PHR1), which is involved in pyrimidine dimer repair. Strikingly, the FEL lost 33 additional genes, including polymerases (i.e., POLymerase 4 [POL4] and POL32) and telomere-associated genes (e.g., Repressor/activator site binding protein-Interacting Factor 1 [RIF1], Replication Factor A 3 [RFA3], Cell Division Cycle 13 [CDC13], Pbp1p Binding Protein [PBP2]). Echoing these losses, molecular evolutionary analyses reveal that, compared to the SEL, the FEL stem lineage underwent a burst of accelerated evolution, which resulted in greater mutational loads, homopolymer instabilities, and higher fractions of mutations associated with the common endogenously damaged base, 8-oxoguanine. We conclude that Hanseniaspora is an ancient lineage that has diversified and thrived, despite lacking many otherwise highly conserved cell-cycle and genome integrity genes and pathways, and may represent a novel, to our knowledge, system for studying cellular life without them.
Asunto(s)
Ciclo Celular/genética , Reparación del ADN/genética , Genes Fúngicos , Filogenia , Saccharomycetales/citología , Saccharomycetales/genética , Secuencia de Bases , Daño del ADN/genética , Evolución Molecular , FenotipoRESUMEN
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.
Asunto(s)
Daño del ADN , ADN Glicosilasas/metabolismo , Reparación del ADN , Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismoRESUMEN
The NEIL3 DNA glycosylase maintains genome integrity during replication by excising oxidized bases from single-stranded DNA (ssDNA) and unhooking interstrand cross-links (ICLs) at fork structures. In addition to its N-terminal catalytic glycosylase domain, NEIL3 contains two tandem C-terminal GRF-type zinc fingers that are absent in the other NEIL paralogs. ssDNA binding by the GRF-ZF motifs helps recruit NEIL3 to replication forks converged at an ICL, but the nature of DNA binding and the effect of the GRF-ZF domain on catalysis of base excision and ICL unhooking is unknown. Here, we show that the tandem GRF-ZFs of NEIL3 provide affinity and specificity for DNA that is greater than each individual motif alone. The crystal structure of the GRF domain shows that the tandem ZF motifs adopt a flexible head-to-tail configuration well-suited for binding to multiple ssDNA conformations. Functionally, we establish that the NEIL3 GRF domain inhibits glycosylase activity against monoadducts and ICLs. This autoinhibitory activity contrasts GRF-ZF domains of other DNA-processing enzymes, which typically use ssDNA binding to enhance catalytic activity, and suggests that the C-terminal region of NEIL3 is involved in both DNA damage recruitment and enzymatic regulation.
Asunto(s)
ADN de Cadena Simple/metabolismo , N-Glicosil Hidrolasas/metabolismo , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , ADN/metabolismo , Replicación del ADN , ADN de Cadena Simple/química , Humanos , Ratones , N-Glicosil Hidrolasas/antagonistas & inhibidores , N-Glicosil Hidrolasas/genética , Unión Proteica , Estructura Terciaria de Proteína , Alineación de Secuencia , Dedos de ZincRESUMEN
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 product complexes. Instead of directly interacting with the damaged nucleobase, AlkD recognizes aberrant base pairs through interactions with the phosphoribose backbone, while the lesion remains stacked in the DNA duplex. Quantum mechanical calculations revealed that these contacts include catalytic charge-dipole and CH-π interactions that preferentially stabilize the transition state. We show in vitro and in vivo how this unique means of recognition and catalysis enables AlkD to repair large adducts formed by yatakemycin, a member of the duocarmycin family of antimicrobial natural products exploited in bacterial warfare and chemotherapeutic trials. Bulky adducts of this or any type are not excised by DNA glycosylases that use a traditional base-flipping mechanism. Hence, these findings represent a new model for DNA repair and provide insights into catalysis of base excision.
Asunto(s)
Bacillus cereus/enzimología , Biocatálisis , Aductos de ADN/química , Aductos de ADN/metabolismo , ADN Glicosilasas/química , ADN Glicosilasas/metabolismo , Reparación del ADN , Emparejamiento Base , Dominio Catalítico , Cristalografía por Rayos X , Daño del ADN , Duocarmicinas , Indoles , Modelos Moleculares , PirrolesRESUMEN
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.
Asunto(s)
Archaea/enzimología , Bacterias/enzimología , ADN Glicosilasas/metabolismo , Reparación del ADN/genética , ADN/metabolismo , Eucariontes/enzimología , Archaea/genética , Bacterias/genética , Cristalografía por Rayos X , Daño del ADN/genética , Eucariontes/genética , Conformación ProteicaRESUMEN
DNA glycosylases are important editing enzymes that protect genomic stability by excising chemically modified nucleobases that alter normal DNA metabolism. These enzymes have been known only to initiate base excision repair of small adducts by extrusion from the DNA helix. However, recent reports have described both vertebrate and microbial DNA glycosylases capable of unhooking highly toxic interstrand cross-links (ICLs) and bulky minor groove adducts normally recognized by Fanconi anemia and nucleotide excision repair machinery, although the mechanisms of these activities are unknown. Here we report the crystal structure of Streptomyces sahachiroi AlkZ (previously Orf1), a bacterial DNA glycosylase that protects its host by excising ICLs derived from azinomycin B (AZB), a potent antimicrobial and antitumor genotoxin. AlkZ adopts a unique fold in which three tandem winged helix-turn-helix motifs scaffold a positively charged concave surface perfectly shaped for duplex DNA. Through mutational analysis, we identified two glutamine residues and a ß-hairpin within this putative DNA-binding cleft that are essential for catalytic activity. Additionally, we present a molecular docking model for how this active site can unhook either or both sides of an AZB ICL, providing a basis for understanding the mechanisms of base excision repair of ICLs. Given the prevalence of this protein fold in pathogenic bacteria, this work also lays the foundation for an emerging role of DNA repair in bacteria-host pathogenesis.
Asunto(s)
Proteínas Bacterianas/metabolismo , ADN Glicosilasas/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Streptomyces/enzimología , Antibacterianos , Proteínas Bacterianas/genética , ADN Glicosilasas/química , ADN Glicosilasas/genética , ADN Bacteriano/química , ADN Bacteriano/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Modelos Moleculares , Mutación , Naftalenos/química , Naftalenos/metabolismo , Péptidos/química , Péptidos/metabolismo , Unión Proteica , Conformación Proteica , Pliegue de ProteínaRESUMEN
SMARCAL1 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A-like1) maintains genome integrity during DNA replication. Here we investigated its mechanism of action. We found that SMARCAL1 travels with elongating replication forks, and its absence leads to MUS81-dependent double-strand break formation. Binding to specific nucleic acid substrates activates SMARCAL1 activity in a reaction that requires its HARP2 (Hep-A-related protein 2) domain. Homology modeling indicates that the HARP domain is similar in structure to the DNA-binding domain of the PUR proteins. Limited proteolysis, small-angle X-ray scattering, and functional assays indicate that the core enzymatic unit consists of the HARP2 and ATPase domains that fold into a stable structure. Surprisingly, SMARCAL1 is capable of binding three-way and four-way Holliday junctions and model replication forks that lack a designed ssDNA region. Furthermore, SMARCAL1 remodels these DNA substrates by promoting branch migration and fork regression. SMARCAL1 mutations that cause Schimke immunoosseous dysplasia or that inactivate the HARP2 domain abrogate these activities. These results suggest that SMARCAL1 continuously surveys replication forks for damage. If damage is present, it remodels the fork to promote repair and restart. Failures in the process lead to activation of an alternative repair mechanism that depends on MUS81-catalyzed cleavage of the damaged fork.
Asunto(s)
ADN Helicasas/metabolismo , Replicación del ADN/fisiología , ADN Cruciforme/metabolismo , Inestabilidad Genómica/fisiología , Línea Celular Tumoral , Roturas del ADN de Doble Cadena , ADN Helicasas/genética , Replicación del ADN/genética , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Endonucleasas/metabolismo , Inestabilidad Genómica/genética , Células HEK293 , Humanos , Unión Proteica , Estructura Terciaria de Proteína , Fase SRESUMEN
Helicase-like transcription factor (HLTF) is a central mediator of the DNA damage response and maintains genome stability by regressing stalled replication forks. The N-terminal HIRAN domain binds specifically to the 3'-end of single-stranded DNA (ssDNA), and disrupting this function interferes with fork regression in vitro as well as replication fork progression in cells under replication stress. Here, we investigated the mechanism by which the HIRAN-ssDNA interaction facilitates fork remodeling. Our results indicated that HIRAN capture of a denatured nascent leading 3'-end directs specific binding of HLTF to forks. DNase footprinting revealed that HLTF binds to the parental duplex ahead of the fork and at the leading edge behind the fork. Moreover, we found that the HIRAN domain is important for initiating regression of forks when both nascent strands are at the junction, but is dispensable when forks contain ssDNA regions on either template strand. We also found that HLTF catalyzes fork restoration from a partially regressed structure in a HIRAN-dependent manner. Thus, HIRAN serves as a substrate-recognition domain to properly orient the ATPase motor domain at stalled and regressed forks and initiates fork remodeling by guiding formation of a four-way junction. We discuss how these activities compare with those of two related fork remodelers, SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A-like 1 (SMARCAL1) and zinc finger RANBP2 type-containing 3 (ZRANB3) to provide insight into their nonredundant roles in DNA damage tolerance.
Asunto(s)
ADN Helicasas/metabolismo , Replicación del ADN , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/metabolismo , ADN Helicasas/química , ADN Helicasas/genética , ADN de Cadena Simple/química , ADN de Cadena Simple/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Humanos , Dominios Proteicos , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
Yatakemycin (YTM) is an extraordinarily toxic DNA alkylating agent with potent antimicrobial and antitumor properties and is the most recent addition to the CC-1065 and duocarmycin family of natural products. Though 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 the way they are eliminated by BER have been unclear. Here, we describe the structural and biochemical properties of YTM adducts that are 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.
Asunto(s)
Productos Biológicos/toxicidad , Aductos de ADN/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Indoles/toxicidad , Pirroles/toxicidad , Productos Biológicos/farmacología , Daño del ADN , Farmacorresistencia Bacteriana , Duocarmicinas , Indoles/farmacología , Estructura Molecular , Pirroles/farmacologíaRESUMEN
In this issue of Molecular Cell, Dango and Mosammaparast discover that the human oxidative demethylase ALKBH3 functions in complex with a DNA helicase to eliminate N3-methylcytosine lesions from ssDNA and that specific cancer cell lines are dependent on this activity for proliferation (Dango et al., 2011).