RESUMEN
DNA polymerases lambda (Polλ) and mu (Polµ) are X-Family polymerases that participate in DNA double-strand break (DSB) repair by the nonhomologous end-joining pathway (NHEJ). Both polymerases direct synthesis from one DSB end, using template derived from a second DSB end. In this way, they promote the NHEJ ligation step and minimize the sequence loss normally associated with this pathway. The two polymerases differ in cognate substrate, as Polλ is preferred when synthesis must be primed from a base-paired DSB end, while Polµ is required when synthesis must be primed from an unpaired DSB end. We generated a Polλ variant (PolλKGET) that retained canonical Polλ activity on a paired end-albeit with reduced incorporation fidelity. We recently discovered that the variant had unexpectedly acquired the activity previously unique to Polµ-synthesis from an unpaired primer terminus. Though the sidechains of the Loop1 region make no contact with the DNA substrate, PolλKGET Loop1 amino acid sequence is surprisingly essential for its unique activity during NHEJ. Taken together, these results underscore that the Loop1 region plays distinct roles in different Family X polymerases.
Asunto(s)
ADN Polimerasa beta , ADN Polimerasa Dirigida por ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Mutación con Ganancia de Función , ADN Polimerasa beta/metabolismo , Reparación del ADN , ADN/metabolismo , Reparación del ADN por Unión de ExtremidadesRESUMEN
DNA double-strand breaks (DSBs) threaten genomic stability, since their persistence can lead to loss of critical genetic information, chromosomal translocations or rearrangements, and cell death. DSBs can be repaired through the nonhomologous end-joining pathway (NHEJ), which processes and ligates DNA ends efficiently to prevent or minimize sequence loss. Polymerase λ (Polλ), one of the Family X polymerases, fills sequence gaps of DSB substrates with a strict specificity for a base-paired primer terminus. There is little information regarding Polλ's approach to engaging such substrates. We used in vitro polymerization and cell-based NHEJ assays to explore the contributions of conserved loop regions toward DSB substrate specificity and utilization. In addition, we present multiple crystal structures of Polλ in synapsis with varying biologically relevant DSB end configurations, revealing how key structural features and hydrogen bonding networks work in concert to stabilize these tenuous, potentially cytotoxic DNA lesions during NHEJ.
Asunto(s)
Emparejamiento Cromosómico , Reparación del ADN por Unión de Extremidades , Roturas del ADN de Doble Cadena , Nucleotidiltransferasas , Especificidad por Sustrato , SinapsisRESUMEN
8-oxo-guanine (8OG) is a common base lesion, generated by reactive oxygen species, which has been associated with human diseases such as cancer, aging-related neurodegenerative disorders and atherosclerosis. 8OG is highly mutagenic, due to its dual-coding potential it can pair both with adenine or cytidine. Therefore, it creates a challenge for DNA polymerases striving to correctly replicate and/or repair genomic or mitochondrial DNA. Numerous structural studies provide insights into the mechanistic basis of the specificity of 8OG bypass by DNA polymerases from different families. Here, we focus on how repair polymerases from Family X (Pols ß, λ and µ) engage DNA substrates containing the oxidized guanine. We review structures of binary and ternary complexes for the three polymerases, which represent distinct steps in their catalytic cycles-the binding of the DNA substrate and the incoming nucleotide, followed by its insertion and extension. At each of these steps, the polymerase may favor or exclude the correct C or incorrect A, affecting the final outcome, which varies depending on the enzyme.
Asunto(s)
8-Hidroxi-2'-Desoxicoguanosina/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Dominio Catalítico/genética , ADN/genética , ADN/metabolismo , Reparación del ADN/genética , Replicación del ADN/genética , ADN Polimerasa Dirigida por ADN/genética , HumanosRESUMEN
DNA polymerase µ is a Family X member that participates in repair of DNA double strand breaks (DSBs) by non-homologous end joining. Its role is to fill short gaps arising as intermediates in the process of V(D)J recombination and during processing of accidental double strand breaks. Pol µ is the only known template-dependent polymerase that can repair non-complementary DSBs with unpaired 3´primer termini. Here we review the unique properties of Pol µ that allow it to productively engage such a highly unstable substrate to generate a nick that can be sealed by DNA Ligase IV.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , ADN Polimerasa Dirigida por ADN/metabolismo , ADN/metabolismo , ADN Ligasa (ATP)/metabolismo , HumanosRESUMEN
Genomic integrity is threatened by cytotoxic DNA double-strand breaks (DSBs), which must be resolved efficiently to prevent sequence loss, chromosomal rearrangements/translocations, or cell death. Polymerase µ (Polµ) participates in DSB repair via the nonhomologous end-joining (NHEJ) pathway, by filling small sequence gaps in broken ends to create substrates ultimately ligatable by DNA Ligase IV. Here we present structures of human Polµ engaging a DSB substrate. Synapsis is mediated solely by Polµ, facilitated by single-nucleotide homology at the break site, wherein both ends of the discontinuous template strand are stabilized by a hydrogen bonding network. The active site in the quaternary Pol µ complex is poised for catalysis and nucleotide incoporation proceeds in crystallo. These structures demonstrate that Polµ may address complementary DSB substrates during NHEJ in a manner indistinguishable from single-strand breaks.
Asunto(s)
Roturas del ADN de Doble Cadena , ADN Polimerasa Dirigida por ADN/química , ADN/química , Cristalografía por Rayos X , Daño del ADN , Reparación del ADN por Unión de Extremidades , ADN Ligasa (ATP)/metabolismo , Reparación del ADN , Proteínas de Unión al ADN/química , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Conformación ProteicaRESUMEN
DNA double-strand breaks (DSBs) resulting from reactive oxygen species generated by exposure to UV and ionizing radiation are characterized by clusters of lesions near break sites. Such complex DSBs are repaired slowly, and their persistence can have severe consequences for human health. We have therefore probed DNA break repair containing a template 8-oxo-7,8-dihydro-2'-guanosine (8OG) by Family X Polymerase µ (Pol µ) in steady-state kinetics and cell-based assays. Pol µ tolerates 8OG-containing template DNA substrates, and the filled products can be subsequently ligated by DNA Ligase IV during Nonhomologous end-joining. Furthermore, Pol µ exhibits a strong preference for mutagenic bypass of 8OG by insertion of adenine. Crystal structures reveal that the template 8OG is accommodated in the Pol µ active site with none of the DNA substrate distortions observed for Family X siblings Pols ß or λ. Kinetic characterization of template 8OG bypass indicates that Pol µ inserts adenosine nucleotides with weak sugar selectivity and, given the high cellular concentration of ATP, likely performs its role in repair of complex 8OG-containing DSBs using ribonucleotides.
Asunto(s)
Roturas del ADN de Doble Cadena/efectos de la radiación , Reparación del ADN por Unión de Extremidades/genética , ADN Polimerasa Dirigida por ADN/genética , Guanosina/análogos & derivados , Adenosina Trifosfato/genética , Daño del ADN/genética , Daño del ADN/efectos de la radiación , Reparación del ADN por Unión de Extremidades/efectos de la radiación , ADN Ligasa (ATP)/genética , Replicación del ADN/genética , ADN Polimerasa Dirigida por ADN/química , Guanosina/genética , Humanos , Mutagénesis/efectos de la radiación , Radiación Ionizante , Especies Reactivas de Oxígeno/química , Rayos UltravioletaRESUMEN
DNA ligase IV (LigIV) performs the final DNA nick-sealing step of classical nonhomologous end-joining, which is critical for immunoglobulin gene maturation and efficient repair of genotoxic DNA double-strand breaks. Hypomorphic LigIV mutations cause extreme radiation sensitivity and immunodeficiency in humans. To better understand the unique features of LigIV function, here we report the crystal structure of the catalytic core of human LigIV in complex with a nicked nucleic acid substrate in two distinct states-an open lysyl-AMP intermediate, and a closed DNA-adenylate form. Results from structural and mutagenesis experiments unveil a dynamic LigIV DNA encirclement mechanism characterized by extensive interdomain interactions and active site phosphoanhydride coordination, all of which are required for efficient DNA nick sealing. These studies provide a scaffold for defining impacts of LigIV catalytic core mutations and deficiencies in human LIG4 syndrome.
Asunto(s)
Biocatálisis , Dominio Catalítico , ADN Ligasa (ATP)/química , ADN Ligasa (ATP)/metabolismo , ADN/metabolismo , Adenina/metabolismo , Secuencia de Bases , ADN Ligasa (ATP)/genética , Humanos , Lisina/metabolismo , Mutagénesis/genética , Mutación/genética , Polimorfismo Genético , Unión Proteica , Especificidad por SustratoRESUMEN
INTRODUCTION: Scleroderma is a chronic connective tissue disease resulting in fibrosis. AIM: The aim of the study was to determine the connection between sE-selectin and sIL-2R and the severity of skin lesions in various subtypes of LoS. Evaluation of disease severity, the location of skin lesions, the duration of symptoms and disease activity were assessed in relation to the three different LoS subtypes in patients with localized scleroderma. MATERIAL AND METHODS: The study included 42 patients with localized scleroderma and the control group consisted of 41 healthy subjects. All patients in the LoS study group had a confirmed diagnosis via skin biopsy and underwent serology testing for sE-selectin and sIL-2R concentrations by enzyme-linked immunosorbent assay (ELISA). RESULTS: Significantly higher levels of sE-selectin and sIL-2R were observed in the LoS study group when compared with the control group (p < 0.001). The analysis showed a result close to statistical significance (p = 0.058) between sE-selectin concentration during the time of active disease in the LoS study group. The highest concentrations of sE-selectin and sIL-2R were observed in patients with the generalized subtype of LoS. A positive, statistically significant, curvilinear relationship was shown amid the modified Localized Skin Severity Index (mLoSSI) and sE-selectin and sIL-2R concentrations in the LoS study group. CONCLUSIONS: Concentrations of the circulating form of sE-selectin appear to be an adequate marker of the endothelial function, positively correlating with the severity of the disease. The proven correlation of sIL-2R concentrations with the severity of the disease indicates that it is a valuable prognostic factor for predicting the impending course of the disease.
RESUMEN
The rapid progress of genetic engineering furthermore opens up new prospects in the therapy of this difficult-to-treat disease. IL-23 inhibitors, phosphodiesterase 4 (PDE4) inhibitors, and Janus kinase (JAK) inhibitors are currently encouraging further research. Two drugs which are IL-23 inhibitors are now in phase III of clinical trials. The aim of the action of both drugs is selective IL-23 inhibition by targeting the p19 subunit. Guselkumab is a fully human monoclonal antibody. Tildrakizumab is a humanized monoclonal antibody, which also belongs to IgG class and is targeted to subunit p19 of interleukin 23 (IL-23). Phosphodiesterase inhibitors exert an anti-inflammatory action and their most common group is the PDE4 family. PDE4 inhibits cAMP, which reduces the inflammatory response of the pathway of Th helper lymphocytes, Th17, and type 1 interferon which modulates the production of anti-inflammatory cytokines such as IL-10 interleukins. The Janus kinase (JAK) signaling pathway plays an important role in the immunopathogenesis of psoriasis. Tofacitinib suppresses the expression of IL-23, IL-17A, IL-17F, and IL-22 receptors during the stimulation of lymphocytes. Ruxolitinib is a selective inhibitor of JAK1 and JAK2 kinases and the JAK-STAT signaling pathway. This article is a review of the aforementioned drugs as described in the latest available literature.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Fármacos Dermatológicos/uso terapéutico , Subunidad p19 de la Interleucina-23/antagonistas & inhibidores , Inhibidores de las Cinasas Janus/uso terapéutico , Quinasas Janus/antagonistas & inhibidores , Inhibidores de Fosfodiesterasa 4/uso terapéutico , Psoriasis/tratamiento farmacológico , Piel/efectos de los fármacos , Animales , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/inmunología , Fármacos Dermatológicos/efectos adversos , Humanos , Subunidad p19 de la Interleucina-23/inmunología , Subunidad p19 de la Interleucina-23/metabolismo , Inhibidores de las Cinasas Janus/efectos adversos , Quinasas Janus/metabolismo , Terapia Molecular Dirigida , Inhibidores de Fosfodiesterasa 4/efectos adversos , Psoriasis/diagnóstico , Psoriasis/enzimología , Psoriasis/inmunología , Transducción de Señal/efectos de los fármacos , Piel/enzimología , Piel/inmunología , Piel/patología , Resultado del TratamientoRESUMEN
While most DNA polymerases discriminate against ribonucleotide triphosphate (rNTP) incorporation very effectively, the Family X member DNA polymerase µ (Pol µ) incorporates rNTPs almost as efficiently as deoxyribonucleotides. To gain insight into how this occurs, here we have used X-ray crystallography to describe the structures of pre- and post-catalytic complexes of Pol µ with a ribonucleotide bound at the active site. These structures reveal that Pol µ binds and incorporates a rNTP with normal active site geometry and no distortion of the DNA substrate or nucleotide. Moreover, a comparison of rNTP incorporation kinetics by wildtype and mutant Pol µ indicates that rNTP accommodation involves synergistic interactions with multiple active site residues not found in polymerases with greater discrimination. Together, the results are consistent with the hypothesis that rNTP incorporation by Pol µ is advantageous in gap-filling synthesis during DNA double strand break repair by nonhomologous end joining, particularly in nonreplicating cells containing very low deoxyribonucleotide concentrations.
Asunto(s)
Reparación del ADN por Unión de Extremidades , ADN Polimerasa Dirigida por ADN/química , ADN/química , Desoxirribonucleótidos/química , Ribonucleótidos/química , Secuencias de Aminoácidos , Secuencia de Bases , Dominio Catalítico , Clonación Molecular , Cristalografía por Rayos X , ADN/metabolismo , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Desoxirribonucleótidos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Humanos , Cinética , Modelos Moleculares , Conformación de Ácido Nucleico , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonucleótidos/metabolismo , Especificidad por Sustrato , TermodinámicaRESUMEN
DNA polymerase (pol) µ is a DNA-dependent polymerase that incorporates nucleotides during gap-filling synthesis in the non-homologous end-joining pathway of double-strand break repair. Here we report time-lapse X-ray crystallography snapshots of catalytic events during gap-filling DNA synthesis by pol µ. Unique catalytic intermediates and active site conformational changes that underlie catalysis are uncovered, and a transient third (product) metal ion is observed in the product state. The product manganese coordinates phosphate oxygens of the inserted nucleotide and PPi. The product metal is not observed during DNA synthesis in the presence of magnesium. Kinetic analyses indicate that manganese increases the rate constant for deoxynucleoside 5'-triphosphate insertion compared to magnesium. The likely product stabilization role of the manganese product metal in pol µ is discussed. These observations provide insight on structural attributes of this X-family double-strand break repair polymerase that impact its biological function in genome maintenance.DNA polymerase (pol) µ functions in DNA double-strand break repair. Here the authors use time-lapse X-ray crystallography to capture the states of pol µ during the conversion from pre-catalytic to product complex and observe a third transiently bound metal ion in the product state.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , ADN/genética , Dominio Catalítico , Cristalografía por Rayos X , ADN/química , ADN/metabolismo , Replicación del ADN , ADN Polimerasa Dirigida por ADN/química , Cinética , Modelos Moleculares , Nucleótidos/metabolismoRESUMEN
BACKGROUND: Hyaluronic acid is a widely available, biocompatible, polysaccharide with distinguishing physiochemical properties which inspire its application throughout several fields of medicine. OBJECTIVE: We aim to investigate the application of hyaluronic acid and its effectiveness throughout several fields of medicine, including several therapies administered and prescribed by general health practitioners. METHODS: We conducted a systematic review on randomized controlled trials about the physiochemical properties of hyaluronic acid and its application through primary care. Studies included in this review were peer reviewed and met our inclusion criteria. FINDINGS: Factors were clustered into the following: uses throughout several fields of medicine, physiochemical properties, bioavailability, tolerance, effectiveness, and adverse effects. Therapies with hyaluronic acid provided long-lasting, pain relieving, moisturizing, lubricating, and dermal filling effect. Tissue hydration, elasticity, and durability improved. CONCLUSIONS: Adjunct therapy with hyaluronic acid provides longer-lasting therapeutic effect when compared to the use of glucocorticosteroids and NSAIDs in osteoarthritic chronic diseases, is well-established in ophthalmology due to its lubricating properties for the corneal endothelium, and improves tissue hydration and cellular resistance to mechanical damage in aesthetic dermatology, and has marginal adverse effects. Several trials indicated its role in tumor markers, liver diseases, and in pharmaceuticals, but further research would be necessary to draw conclusive results in those fields.
Asunto(s)
Rellenos Dérmicos/uso terapéutico , Ácido Hialurónico/uso terapéutico , Neoplasias/metabolismo , Viscosuplementos/uso terapéutico , Disponibilidad Biológica , Técnicas Cosméticas , Rellenos Dérmicos/efectos adversos , Rellenos Dérmicos/farmacocinética , Síndromes de Ojo Seco/tratamiento farmacológico , Humanos , Ácido Hialurónico/efectos adversos , Ácido Hialurónico/metabolismo , Ácido Hialurónico/farmacocinética , Osteoartritis/tratamiento farmacológico , Ensayos Clínicos Controlados Aleatorios como Asunto , Viscosuplementos/efectos adversos , Viscosuplementos/farmacocinéticaRESUMEN
Among the many proteins used to repair DNA double-strand breaks by nonhomologous end joining (NHEJ) are two related family X DNA polymerases, Pol λ and Pol µ. Which of these two polymerases is preferentially used for filling DNA gaps during NHEJ partly depends on sequence complementarity at the break, with Pol λ and Pol µ repairing complementary and noncomplementary ends, respectively. To better understand these substrate preferences, we present crystal structures of Pol µ on a 2-nt gapped DNA substrate, representing three steps of the catalytic cycle. In striking contrast to Pol λ, Pol µ "skips" the first available template nucleotide, instead using the template base at the 5' end of the gap to direct nucleotide binding and incorporation. This remarkable divergence from canonical 3'-end gap filling is consistent with data on end-joining substrate specificity in cells, and provides insights into polymerase substrate choices during NHEJ.
Asunto(s)
Reparación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , ADN/biosíntesis , Catálisis , Cristalografía por Rayos X , Daño del ADN , ADN Polimerasa beta/química , Humanos , Cinética , Conformación de Ácido Nucleico , Nucleótidos/genética , Estructura Secundaria de Proteína , Análisis de Secuencia de ADN , Especificidad por SustratoRESUMEN
PrimPol is a recently described DNA polymerase that has the virtue of initiating DNA synthesis. In addition of being a sensu stricto DNA primase, PrimPol's polymerase activity has a large capacity to tolerate different kind of lesions. The different strategies used by PrimPol for DNA damage tolerance are based on its capacity to "read" certain lesions, to skip unreadable lesions, and as an ultimate solution, to restart DNA synthesis beyond the lesion thus acting as a TLS primase. This lesion bypass potential, revised in this article, is strengthened by the preferential use of moderate concentrations of manganese ions as the preferred metal activator. We show here that PrimPol is able to extend RNA primers with ribonucleotides, even when bypassing 8oxoG lesions, suggesting a potential new scenario for PrimPol as a TLS polymerase assisting transcription. We also show that PrimPol displays a high degree of versatility to accept or induce distortions of both primer and template strands, creating alternative alignments based on microhomology that would serve to skip unreadable lesions and to connect separate strands. In good agreement, PrimPol is highly prone to generate indels at short nucleotide repeats. Finally, an evolutionary view of the relationship between translesion synthesis and primase functions is briefly discussed.
Asunto(s)
ADN Primasa/metabolismo , Reparación del ADN , Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , ADN/metabolismo , Enzimas Multifuncionales/metabolismo , Cationes , ADN/biosíntesis , Daño del ADN , ADN Primasa/química , ADN Polimerasa Dirigida por ADN/química , Humanos , Manganeso/química , Enzimas Multifuncionales/química , Conformación de Ácido NucleicoRESUMEN
DNA polymerase λ (pol λ) functions in DNA repair with its main roles considered to be filling short gaps during repair of double-strand breaks by nonhomologous end joining and during base excision repair. As indicated by structural and biochemical studies over the past 10 years, pol λ shares many common properties with other family X siblings (pol ß, pol µ, and terminal deoxynucleotidyl transferase) but also has unique structural features that determine its specific functions. In this review, we consider how structural studies over the past decade furthered our understanding of the behavior and biological roles of pol λ.
Asunto(s)
ADN Polimerasa beta/química , ADN Polimerasa beta/metabolismo , Reparación del ADN/fisiología , Catálisis , Reparación del ADN por Unión de Extremidades/fisiología , ADN Polimerasa beta/genética , Desoxirribonucleótidos/metabolismo , Modelos Moleculares , Conformación Proteica , Estructura Terciaria de Proteína/fisiología , Ribonucleótidos/metabolismoRESUMEN
Protozoans of the genus Leishmania, the pathogenic agent causing leishmaniasis, encode the family X DNA polymerase Li Pol ß. Here, we report the first crystal structures of Li Pol ß. Our pre- and post-catalytic structures show that the polymerase adopts the common family X DNA polymerase fold. However, in contrast to other family X DNA polymerases, the dNTP-induced conformational changes in Li Pol ß are much more subtle. Moreover, pre- and post-catalytic structures reveal that Li Pol ß interacts with the template strand through a nonconserved, variable region known as loop3. Li Pol ß Δloop3 mutants display a higher catalytic rate, catalytic efficiency and overall error rates with respect to WT Li Pol ß. These results further demonstrate the subtle structural variability that exists within this family of enzymes and provides insight into how this variability underlies the substantial functional differences among their members.
Asunto(s)
Dominio Catalítico , ADN Polimerasa beta/química , ADN Polimerasa beta/genética , Leishmania infantum/enzimología , Cristalografía por Rayos X , ADN Polimerasa beta/metabolismo , Modelos Moleculares , Mutación , Conformación Proteica , Pliegue de Proteína , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
DNA polymerase µ (Pol µ) is the only template-dependent human DNA polymerase capable of repairing double-strand DNA breaks (DSBs) with unpaired 3' ends in nonhomologous end joining (NHEJ). To probe this function, we structurally characterized Pol µ's catalytic cycle for single-nucleotide incorporation. These structures indicate that, unlike other template-dependent DNA polymerases, Pol µ shows no large-scale conformational changes in protein subdomains, amino acid side chains or DNA upon dNTP binding or catalysis. Instead, the only major conformational change is seen earlier in the catalytic cycle, when the flexible loop 1 region repositions upon DNA binding. Pol µ variants with changes in loop 1 have altered catalytic properties and are partially defective in NHEJ. The results indicate that specific loop 1 residues contribute to Pol µ's unique ability to catalyze template-dependent NHEJ of DSBs with unpaired 3' ends.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN , ADN Polimerasa Dirigida por ADN/química , Catálisis , Dominio Catalítico , Cristalización , Cristalografía por Rayos X , ADN Polimerasa Dirigida por ADN/genética , Electrones , Humanos , Cinética , Modelos Moleculares , Mutación , Nucleótidos/química , Unión Proteica , Especificidad por SustratoRESUMEN
Base excision repair (BER) plays a vital role in maintaining genomic integrity in mammalian cells. DNA polymerase λ (Pol λ) is believed to play a backup role to DNA polymerase ß (Pol ß) in base excision repair. Two oxidized abasic lesions that are produced by a variety of DNA-damaging agents, including several antitumor antibiotics, the C4'-oxidized abasic site following Ape1 incision (pC4-AP), and 5'-(2-phosphoryl-1,4-dioxobutane) (DOB), irreversibly inactivate Pol ß and Pol λ. The interactions of DOB and pC4-AP with Pol λ are examined in detail using DNA substrates containing these lesions at defined sites. Single-turnover kinetic experiments show that Pol λ excises DOB almost 13 times more slowly than a 5'-phosphorylated 2-deoxyribose (dRP). pC4-AP is excised approximately twice as fast as DOB. The absolute rate constants are considerably slower than those reported for Pol ß for the respective reactions, suggesting that Pol λ may be an inefficient backup in BER. DOB inactivates Pol λ approximately 3-fold less efficiently than it does Pol ß, and the difference can be attributed to a higher K(I) (33 ± 7 nM). Inactivation of Pol λ's lyase activity by DOB also prevents the enzyme from conducting polymerization following preincubation of the protein and DNA. Mass spectral analysis of GluC-digested Pol λ inactivated by DOB shows that Lys324 is modified. There is inferential support for the idea that Lys312 may also be modified. Both residues are within the Pol λ lyase active site. When acting on pC4-AP, Pol λ achieves approximately four turnovers on average before being inactivated. Lyase inactivation by pC4-AP is also accompanied by loss of polymerase activity, and mass spectrometry indicates that Lys312 and Lys324 are modified by the lesion. The ability of DOB and pC4-AP to inactivate Pol λ provides additional evidence that these lesions are significant sources of the cytotoxicity of DNA-damaging agents that produce them.
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Butanonas/metabolismo , ADN Polimerasa beta/metabolismo , ADN/química , Desoxirribosa/análogos & derivados , Secuencia de Bases , Butanonas/química , ADN/genética , ADN/metabolismo , Daño del ADN , Desoxirribosa/metabolismo , Activación Enzimática , Humanos , Oxidación-ReducciónRESUMEN
Although most DNA polymerases discriminate against ribonucleotide triphosphaets (rNTPs) during DNA synthesis, recent studies have shown that large numbers of ribonucleotides are incorporated into the eukaryotic nuclear genome. Here, we investigate how a DNA polymerase can stably incorporate an rNTP. The X-ray crystal structure of a variant of human DNA polymerase λ reveals that the rNTP occupies the nucleotide binding pocket without distortion of the active site, despite an unfavorable interaction between the 2'-O and Tyr505 backbone carbonyl. This indicates an energetically unstable binding state for the rNTP, stabilized by additional protein-nucleotide interactions. Supporting this idea is the 200-fold lower catalytic efficiency for rNTP relative to deoxyribonucleotide triphosphate (dNTP) incorporation, reflecting a higher apparent Km value for the rNTP. Furthermore, distortion observed in the structure of the post-catalytic product complex suggests that once the bond between the α- and ß-phosphates of the rNTP is broken, the unfavorable binding state of the ribonucleotide cannot be maintained. Finally, structural and biochemical evaluation of dNTP insertion onto an ribonucleotide monophosphate (rNMP)-terminated primer indicates that a primer-terminal rNMP does not impede extension. The results are relevant to how ribonucleotides are incorporated into DNA in vivo, during replication and during repair, perhaps especially in non-proliferating cells when rNTP:dNTP ratios are high.
Asunto(s)
ADN Polimerasa beta/química , Ribonucleótidos/química , Biocatálisis , Dominio Catalítico , Cristalografía por Rayos X , ADN Polimerasa beta/metabolismo , Humanos , Cinética , Modelos Moleculares , Ribonucleótidos/metabolismoRESUMEN
To test the hypothesis that DNA polymerase ζ participates in Ig hypermutation, we generated two mouse models of Pol ζ function: a B cell-specific conditional knockout and a knock-in strain with a Pol ζ mutagenesis-enhancing mutation. Pol ζ-deficient B cells had a reduction in mutation frequency at Ig loci in the spleen and in Peyer's patches, whereas knock-in mice with a mutagenic Pol ζ displayed a marked increase in mutation frequency in Peyer's patches, revealing a pattern that was similar to mutations in yeast strains with a homologous mutation in the gene encoding the catalytic subunit of Pol ζ. Combined, these data are best explained by a direct role for DNA polymerase ζ in Ig hypermutation.