Structure and mechanism of human DNA polymerase η

The variant form of the human syndrome xeroderma pigmentosum (XPV) is caused by a deficiency in DNA polymerase eta (Pol eta), a DNA polymerase that enables replication through ultraviolet-induced pyrimidine dimers. Here we report high-resolution crystal structures of human Pol eta at four consecutive steps during DNA synthesis through cis-syn cyclobutane thymine dimers. Pol eta acts like a 'molecular splint' to stabilize damaged DNA in a normal B-form conformation. An enlarged active site accommodates the thymine dimer with excellent stereochemistry for two-metal ion catalysis. Two residues conserved among Pol eta orthologues form specific hydrogen bonds with the lesion and the incoming nucleotide to assist translesion synthesis. On the basis of the structures, eight Pol eta missense mutations causing XPV can be rationalized as undermining the molecular splint or perturbing the active-site alignment. The structures also provide an insight into the role of Pol eta in replicating through D loop and DNA fragile sites

Mismatched dNTP incorporation by DNA polymerase β does not proceed via globally different conformational pathways†

Understanding how DNA polymerases control fidelity requires elucidation of the mechanisms of matched and mismatched dNTP incorporations. Little is known about the latter because mismatched complexes do not crystallize readily. In this report, we employed small-angle X-ray scattering (SAXS) and structural modeling to probe the conformations of different intermediate states of mammalian DNA polymerase β (Pol β) in its wild-type and an error-prone variant, I260Q. Our structural results indicate that the mismatched ternary complex lies in-between the open and the closed forms, but more closely resembles the open form for WT and the closed form for I260Q. On the basis of molecular modeling, this over-stabilization of mismatched ternary complex of I260Q is likely caused by formation of a hydrogen bonding network between the side chains of Gln260, Tyr296, Glu295 and Arg258, freeing up Asp192 to coordinate MgdNTP. These results argue against recent reports suggesting that mismatched dNTP incorporations follow a conformational path distinctly different from that of matched dNTP incorporation, or that its conformational closing is a major contributor to fidelity

A nucleotide binding rectification Brownian ratchet model for translocation of Y-family DNA polymerases

Y-family DNA polymerases are characterized by low-fidelity synthesis on undamaged DNA and ability to catalyze translesion synthesis over the damaged DNA. Their translocation along the DNA template is an important event during processive DNA synthesis. In this work we present a Brownian ratchet model for this translocation, where the directed translocation is rectified by the nucleotide binding to the polymerase. Using the model, different features of the available structures for Dpo4, Dbh and polymerase ι in binary and ternary forms can be easily explained. Other dynamic properties of the Y-family polymerases such as the fast translocation event upon dNTP binding for Dpo4 and the considerable variations of the processivity among the polymerases can also be well explained by using the model. In addition, some predicted results of the DNA synthesis rate versus the external force acting on Dpo4 and Dbh polymerases are presented. Moreover, we compare the effect of the external force on the DNA synthesis rate of the Y-family polymerase with that of the replicative DNA polymerase

Tumour suppressor ING1b maintains genomic stability upon replication stress

The lesion bypass pathway, which is regulated by monoubiquitination of proliferating cell nuclear antigen (PCNA), is essential for resolving replication stalling due to DNA lesions. This process is important for preventing genomic instability and cancer development. Previously, it was shown that cells deficient in tumour suppressor p33ING1 (ING1b) are hypersensitive to DNA damaging agents via unknown mechanism. In this study, we demonstrated a novel tumour suppressive function of ING1b in preserving genomic stability upon replication stress through regulating PCNA monoubiquitination. We found that ING1b knockdown cells are more sensitive to UV due to defects in recovering from UV-induced replication blockage, leading to enhanced genomic instability. We revealed that ING1b is required for the E3 ligase Rad18-mediated PCNA monoubiquitination in lesion bypass. Interestingly, ING1b-mediated PCNA monoubiquitination is associated with the regulation of histone H4 acetylation. Results indicate that chromatin remodelling contributes to the stabilization of stalled replication fork and to the regulation of PCNA monoubiquitination during lesion bypass

Steady-state and pseudo-steady-state photocrystallographic studies on linkage isomers of [Ni(Et₄dien)(η²-O,ON)(η¹-NO₂)]:Identification of a new linkage isomer

At temperatures below 150 K, the photoactivated metastable endo‐nitrito linkage isomer [Ni(Et4dien)(η2‐O,ON)(η1‐ONO)] (Et4dien=N,N,N′,N′‐tetraethyldiethylenetriamine) can be generated with 100 % conversion from the ground state nitro‐(η1‐NO2) isomer on irradiation with 500 nm light, in the single crystal by steady‐state photocrystallographic techniques. Kinetic studies show the system is no longer metastable above 150 K, decaying back to the ground state nitro‐(η1‐NO2) arrangement over several hours at 150 K. Variable‐temperature kinetic measurements in the range of 150–160 K show that the rate of endo‐nitrito decay is highly dependent on temperature, and an activation energy of Eact=+48.6(4) kJ mol−1 is calculated for the decay process. Pseudo‐steady‐state experiments, where the crystal is continually pumped by the light source for the duration of the X‐ray experiment, show the production of a previously unobserved, exo‐nitrito‐(η1‐ONO) linkage isomer only at temperatures close to the metastable limit (ca. 140–190 K). This exo isomer is considered to be a transient excited‐state species, as it is only observed in data collected by pseudo‐steady‐state methods

Global Conformational Dynamics of a Y-Family DNA Polymerase during Catalysis

High-resolution analysis of protein, and DNA conformational changes during DNA polymerization, established relationships between the enzymatic function and conformational dynamics of individual domains for a DNA polymerase

Mutations at the Subunit Interface of Yeast Proliferating Cell Nuclear Antigen Reveal a Versatile Regulatory Domain

Acknowledgments We thank Szilvia Minorits for technical assistance. I.U. conceived and designed the project and wrote the manuscript. All authors participated in designing and performing the experiments, and analyzing the results. The authors declare no competing financial interests. This work was also supported by a grant from the National Research, Development and Innovation Office GINOP-2.3.2-15-2016-00001. Funding: This work was supported by Hungarian Science Foundation Grant OTKA 109521 and National Research Development and Innovation Office GINOP-2.3.2-15-2016-00001. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

The RAGNYA fold: a novel fold with multiple topological variants found in functionally diverse nucleic acid, nucleotide and peptide-binding proteins

Using sensitive structure similarity searches, we identify a shared α+β fold, RAGNYA, principally involved in nucleic acid, nucleotide or peptide interactions in a diverse group of proteins. These include the Ribosomal proteins L3 and L1, ATP-grasp modules, the GYF domain, DNA-recombination proteins of the NinB family from caudate bacteriophages, the C-terminal DNA-interacting domain of the Y-family DNA polymerases, the uncharacterized enzyme AMMECR1, the siRNA silencing repressor of tombusviruses, tRNA Wybutosine biosynthesis enzyme Tyw3p, DNA/RNA ligases and related nucleotidyltransferases and the Enhancer of rudimentary proteins. This fold exhibits three distinct circularly permuted versions and is composed of an internal repeat of a unit with two-strands and a helix. We show that despite considerable structural diversity in the fold, its representatives show a common mode of nucleic acid or nucleotide interaction via the exposed face of the sheet. Using this information and sensitive profile-based sequence searches: (1) we predict the active site, and mode of substrate interaction of the Wybutosine biosynthesis enzyme, Tyw3p, and a potential catalytic role for AMMECR1. (2) We provide insights regarding the mode of nucleic acid interaction of the NinB proteins, and the evolution of the active site of classical ATP-grasp enzymes and DNA/RNA ligases. (3) We also present evidence for a bacterial origin of the GYF domain and propose how this version of the fold might have been utilized in peptide interactions in the context of nucleoprotein complexes

Palynological study at the Pliensbachian-Toarcian passage beds of the Rambla del Salto section (Sierra Palomera, Teruel province, Spain)

[ES] El estudio palinológico de los materiales correspondientes al tránsito Pliensbachiense/ Toarciense en la Rambla del Salto (Sierra Palomera, Teruel, España), ha permitido identificar veintiún taxones pertenecientes a distintos grupos de protoctistas, criptógamas vasculares y gimnospermas. Por el momento, ningu- 110 de ellos ha resultado ser relevante desde el punto de vista bloestratigráfico. Se han distinguido dos intervalos caracterizados por su riqueza en elementos planctónicos, cuyas asociaciones palinológicas son de difícil comparación con las de otras cuencas europeas. En los materiales del Toarciense inferior, a partir de las muestras obtenidas en sedimentos del tránsito entre las Subzonas Mirabile y Semicelaíum, tiene lugar la desaparición de los taxones planetónicos y ello podría estar relacionado con el evento subóxico reconocido en otros puntos de la cuenca.[EN] A palynological study carried out at the Pliensbachian-Toarcian passage beds of the Rambla del Salto section (Teruel province, E. Spain) has led to the identification of twenty-one taxa belonging to sorne different groups of Protoctista, gymnospenn and vascular criptogamme plants. The identified taxa show a wide stratigraphic range and have low biostratigraphic value. Two separate intervals are distinguished by their content in planktic elements. In both intervals palynologic associations are difficult to compare with other european basins. Recorded planktic taxa disappear at the Miíabile-Semicelatum Subzone transition (lower Toarcian). This might be related with the suboxie events which has been recognised elsewhere in some other areas in the basin.Este trabajo ha sido financiado por el Proyecto PB93-0459 de la DGICYT. Los autores quieren agradecer al Dr. A. Goy y a la Dra. C. Herrero del Depto. de Paleontología de la Facultad de Ciencias Geológicas de la Universidad Complutense de Madrid la lectura crítica del trabajo, ya que sus observaclones han permitido mejorar el manuscrito original; asf como a los revisores Dr. Dénise Pons y Dr. Javier Ferrer, cuyas opiniones han enriquecido notablemente el trabajo, y al Dr. Guillermo Meléndez por sus correcciones. Las fotografías han sido realizadas por Eulogio Martín Castellanos. úPeer reviewe

Las Hoyas palynology, state of the art

Trabajo presentado en el II International Symposium on Lithographic Limestones.The palynologic study of Las Hoyas lithographic limestone assemblages is a contribution for a general pelobotanic study. The targets of this study are contributions to increase the age determination to integrate pelobotanic and peleoecologic studies.Peer reviewe