Automated AFM analysis of DNA bending reveals initial lesion sensing strategies of DNA glycosylases

Abstract Base excision repair is the dominant DNA repair pathway of chemical modifications such as deamination, oxidation, or alkylation of DNA bases, which endanger genome integrity due to their high mutagenic potential. Detection and excision of these base lesions is achieved by DNA glycosylases. To investigate the remarkably high efficiency in target site search and recognition by these enzymes, we applied single molecule atomic force microscopy (AFM) imaging to a range of glycosylases with structurally different target lesions. Using a novel, automated, unbiased, high-throughput analysis approach, we were able to resolve subtly different conformational states of these glycosylases during DNA lesion search. Our results lend support to a model of enhanced lesion search efficiency through initial lesion detection based on altered mechanical properties at lesions. Furthermore, its enhanced sensitivity and easy applicability also to other systems recommend our novel analysis tool for investigations of diverse, fundamental biological interactions.

Download Full-text

Simple nanofluidic devices for high-throughput, non-equilibrium studies at the single-molecule level

10.1101/201079 ◽

2017 ◽

Author(s):

Carel Fijen ◽

Mattia Fontana ◽

Serge G. Lemay ◽

Klaus Mathwig ◽

Johannes Hohlbein

Keyword(s):

High Throughput ◽

Single Molecule ◽

Single Particle Tracking ◽

Biological Interactions ◽

Dynamic Heterogeneity ◽

High Throughput Analysis ◽

Equilibrium Studies ◽

Single Molecule Level ◽

Nanofluidic Devices ◽

And Performance

ABSTRACTSingle-molecule detection schemes offer powerful means to overcome static and dynamic heterogeneity inherent to complex samples. Probing chemical and biological interactions and reactions with high throughput and time resolution, however, remains challenging and often requires surface-immobilized entities. Here, utilizing camera-based fluorescence microscopy, we present glass-made nanofluidic devices in which fluorescently labelled molecules flow through nanochannels that confine their diffusional movement. The first design features an array of parallel nanochannels for high-throughput analysis of molecular species under equilibrium conditions allowing us to record 200.000 individual localization events in just 10 minutes. Using these localizations for single particle tracking, we were able to obtain accurate flow profiles including flow speeds and diffusion coefficients inside the channels.A second design featuring a T-shaped nanochannel enables precise mixing of two different species as well as the continuous observation of chemical reactions. We utilized the design to visualize enzymatically driven DNA synthesis in real time and at the single-molecule level. Based on our results, we are convinced that the versatility and performance of the nanofluidic devices will enable numerous applications in the life sciences.

Download Full-text

Non-flipping DNA glycosylase AlkD scans DNA without formation of a stable interrogation complex

Communications Biology ◽

10.1038/s42003-021-02400-x ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Arash Ahmadi ◽

Katharina Till ◽

Paul Hoff Backe ◽

Pernille Blicher ◽

Robin Diekmann ◽

...

Keyword(s):

Single Molecule ◽

Excision Repair ◽

Dna Glycosylase ◽

Loss Of Function ◽

Dna Glycosylases ◽

Single Molecule Tracking ◽

Vast Number ◽

Repair Enzymes ◽

Base Excision ◽

Positively Charged

AbstractThe multi-step base excision repair (BER) pathway is initiated by a set of enzymes, known as DNA glycosylases, able to scan DNA and detect modified bases among a vast number of normal bases. While DNA glycosylases in the BER pathway generally bend the DNA and flip damaged bases into lesion specific pockets, the HEAT-like repeat DNA glycosylase AlkD detects and excises bases without sequestering the base from the DNA helix. We show by single-molecule tracking experiments that AlkD scans DNA without forming a stable interrogation complex. This contrasts with previously studied repair enzymes that need to flip bases into lesion-recognition pockets and form stable interrogation complexes. Moreover, we show by design of a loss-of-function mutant that the bimodality in scanning observed for the structural homologue AlkF is due to a key structural differentiator between AlkD and AlkF; a positively charged β-hairpin able to protrude into the major groove of DNA.

Download Full-text

Direct Read and Quantify Damage Nucleotide from an Oligonucleotide Using a Single Molecule Interface

10.26434/chemrxiv.10080638.v2 ◽

2019 ◽

Author(s):

Jiajun Wang ◽

Meng-Yin Li ◽

Jie Yang ◽

Ya-Qian Wang ◽

Xue-Yuan Wu ◽

...

Keyword(s):

Dna Sequencing ◽

Single Molecule ◽

Genetic Diseases ◽

High Sensitivity ◽

Dna Lesions ◽

Lesion Detection ◽

The Novel ◽

Structural Variations ◽

Dna Lesion ◽

Base Lesion

DNA lesion such as metholcytosine(mC), 8-OXO-guanine(OG), inosine(I) etc could cause the genetic diseases. Identification of the varieties of lesion bases are usually beyond the capability of conventional DNA sequencing which is mainly designed to discriminate four bases only. Therefore, lesion detection remain challenge due to the massive varieties and less distinguishable readouts for minor structural variations. Moreover, standard amplification and labelling hardly works in DNA lesions detection. Herein, we designed a single molecule interface from the mutant K238Q Aerolysin, whose confined sensing region shows the high compatible to capture and then directly convert each base lesion into distinguishable current readouts. Compared with previous single molecule sensing interface, the resolution of the K238Q Aerolysin nanopore is enhanced by 2-order. The novel K238Q could direct discriminate at least 3 types (mC, OG, I) lesions without lableing and quantify modification sites under mixed hetero-composition condition of oligonucleotide. Such nanopore could be further applied to diagnose genetic diseases at high sensitivity.

Download Full-text

Mass Spectrometry for Proteomics and Recent Developments in ESI, MALDI and other Ionization Methodologies

Current Proteomics ◽

10.2174/1570164616666190204154653 ◽

2019 ◽

Vol 16 (4) ◽

pp. 267-276

Author(s):

Qurat ul Ain Farooq ◽

Noor ul Haq ◽

Abdul Aziz ◽

Sara Aimen ◽

Muhammad Inam ul Haq

Keyword(s):

Mass Spectrometry ◽

Electrospray Ionization ◽

New Technologies ◽

Protein Complexes ◽

Imaging Techniques ◽

Desorption Electrospray Ionization ◽

High Throughput Analysis ◽

Enhanced Sensitivity ◽

Recent Developments ◽

The Way

Background: Mass spectrometry is a tool used in analytical chemistry to identify components in a chemical compound and it is of tremendous importance in the field of biology for high throughput analysis of biomolecules, among which protein is of great interest. Objective: Advancement in proteomics based on mass spectrometry has led the way to quantify multiple protein complexes, and proteins interactions with DNA/RNA or other chemical compounds which is a breakthrough in the field of bioinformatics. Methods: Many new technologies have been introduced in electrospray ionization (ESI) and Matrixassisted Laser Desorption/Ionization (MALDI) techniques which have enhanced sensitivity, resolution and many other key features for the characterization of proteins. Results: The advent of ambient mass spectrometry and its different versions like Desorption Electrospray Ionization (DESI), DART and ELDI has brought a huge revolution in proteomics research. Different imaging techniques are also introduced in MS to map proteins and other significant biomolecules. These drastic developments have paved the way to analyze large proteins of >200kDa easily. Conclusion: Here, we discuss the recent advancement in mass spectrometry, which is of great importance and it could lead us to further deep analysis of the molecules from different perspectives and further advancement in these techniques will enable us to find better ways for prediction of molecules and their behavioral properties.

Download Full-text

Effect of DNA Glycosylases OGG1 and Neil1 on Oxidized G-Rich Motif in the KRAS Promoter

International Journal of Molecular Sciences ◽

10.3390/ijms22031137 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1137

Author(s):

Annalisa Ferino ◽

Luigi E. Xodo

Keyword(s):

Oxidative Stress ◽

Base Excision Repair ◽

Excision Repair ◽

Start Site ◽

Dna Glycosylases ◽

Repair Pathway ◽

Transcription Start ◽

G Quadruplex ◽

Base Excision ◽

Regulatory Functions

The promoter of the Kirsten ras (KRAS) proto-oncogene contains, upstream of the transcription start site, a quadruplex-forming motif called 32R with regulatory functions. As guanine under oxidative stress can be oxidized to 8-oxoguanine (8OG), we investigated the capacity of glycosylases 8-oxoguanine glycosylase (OGG1) and endonuclease VIII-like 1 (Neil1) to excise 8OG from 32R, either in duplex or G-quadruplex (G4) conformation. We found that OGG1 efficiently excised 8OG from oxidized 32R in duplex but not in G4 conformation. By contrast, glycosylase Neil1 showed more activity on the G4 than the duplex conformation. We also found that the excising activity of Neil1 on folded 32R depended on G4 topology. Our data suggest that Neil1, besides being involved in base excision repair pathway (BER), could play a role on KRAS transcription.

Download Full-text

Highly Sensitive Detection of Paraquat with Pillar[5]arene as an aptamer in α-Hemolysin Nanopore

Materials Chemistry Frontiers ◽

10.1039/d1qm00875g ◽

2021 ◽

Author(s):

Xiaojia Jiang ◽

Mingsong Zang ◽

Fei Li ◽

Chunxi Hou ◽

Quan Luo ◽

...

Keyword(s):

Real Time ◽

High Throughput ◽

Single Molecule ◽

Single Molecule Detection ◽

Sensitive Detection ◽

High Throughput Analysis ◽

Throughput Analysis ◽

The Real ◽

Highly Sensitive ◽

Highly Sensitive Detection

Biological nanopore-based techniques have attracted more and more attention recently in the field of single-molecule detection, because they allow the real-time, sensitive, high-throughput analysis. Herein, we report an engineered biological...

Download Full-text

Recognition of a Tandem Lesion by DNA Glycosylases Explored Combining Molecular Dynamics and Machine Learning

10.1101/2021.01.06.425536 ◽

2021 ◽

Author(s):

Emmanuelle Bignon ◽

Natacha Gillet ◽

Chen-Hui Chan ◽

Tao Jiang ◽

Antonio Monari ◽

...

Keyword(s):

Machine Learning ◽

Molecular Dynamics ◽

Excision Repair ◽

Double Helix ◽

Radiation Chemistry ◽

Dna Lesions ◽

Dynamics Simulation ◽

Interaction Patterns ◽

Abasic Site ◽

Dna Glycosylases

ABSTRACTThe combination of several closely spaced DNA lesions, which can be induced by a single radical hit, constitutes a hallmark in the DNA damage landscape and radiation chemistry. The occurrence of such tandem base lesions give rise to a strong coupling with the double helix degrees of freedom and induce important structural deformations, in contrast to DNA strands containing a single oxidized nucleobase. Although such complex lesions are known to be refractory to repair by DNA glycosylases, there is still a lack of structural evidence to rationalize these phenomena. In this contribution, we explore, by numerical modeling and molecular simulations, the behavior of the bacterial glycosylase responsible for base excision repair (MutM), specialized in excising oxidatively-damaged defects such as 7,8-dihydro-8-oxoguanine (8-oxoG). The difference in lesion recognition between a simple damage and a tandem lesions featuring an additional abasic site is assessed at atomistic resolution owing to microsecond molecular dynamics simulation and machine learning postprocessing, allowing to extensively pinpoint crucial differences in the interaction patterns of the damaged bases. This work advocates for the use of such high throughput numerical simulations for exploring the complex combinatorial chemistry of tandem DNA lesions repair and more generally multiple damaged sites of the utmost significance in radiation chemistry.

Download Full-text

Stochastically pumped adaptation and directional motion of molecular machines

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1714498115 ◽

2018 ◽

Vol 115 (38) ◽

pp. 9405-9413 ◽

Cited By ~ 16

Author(s):

R. Dean Astumian

Keyword(s):

Single Molecule ◽

Molecular Motors ◽

High Efficiency ◽

Probability Distributions ◽

Molecular Machines ◽

High Energy ◽

Stochastic Thermodynamics ◽

Recent Developments ◽

Directional Motion ◽

External Fluctuations

Recent developments in synthetic molecular motors and pumps have sprung from a remarkable confluence of experiment and theory. Synthetic accomplishments have facilitated the ability to design and create molecules, many of them featuring mechanically bonded components, to carry out specific functions in their environment—walking along a polymeric track, unidirectional circling of one ring about another, synthesizing stereoisomers according to an external protocol, or pumping rings onto a long rod-like molecule to form and maintain high-energy, complex, nonequilibrium structures from simpler antecedents. Progress in the theory of nanoscale stochastic thermodynamics, specifically the generalization and extension of the principle of microscopic reversibility to the single-molecule regime, has enhanced the understanding of the design requirements for achieving strong unidirectional motion and high efficiency of these synthetic molecular machines for harnessing energy from external fluctuations to carry out mechanical and/or chemical functions in their environment. A key insight is that the interaction between the fluctuations and the transition state energies plays a central role in determining the steady-state concentrations. Kinetic asymmetry, a requirement for stochastic adaptation, occurs when there is an imbalance in the effect of the fluctuations on the forward and reverse rate constants. Because of strong viscosity, the motions of the machine can be viewed as mechanical equilibrium processes where mechanical resonances are simply impossible but where the probability distributions for the state occupancies and trajectories are very different from those that would be expected at thermodynamic equilibrium.

Download Full-text

Molecular cloning and biological characterization of the human excision repair gene ERCC-3

Molecular and Cellular Biology ◽

10.1128/mcb.10.6.2570-2581.1990 ◽

1990 ◽

Vol 10 (6) ◽

pp. 2570-2581

Author(s):

G Weeda ◽

R C van Ham ◽

R Masurel ◽

A Westerveld ◽

H Odijk ◽

...

Keyword(s):

High Efficiency ◽

Excision Repair ◽

Alkylating Agents ◽

Chinese Hamster ◽

Complementation Group ◽

Cdna Clones ◽

Chromosomal Dna ◽

Dominant Marker ◽

Mammalian Expression ◽

Group 3

In this report we present the cloning, partial characterization, and preliminary studies of the biological activity of a human gene, designated ERCC-3, involved in early steps of the nucleotide excision repair pathway. The gene was cloned after genomic DNA transfection of human (HeLa) chromosomal DNA together with dominant marker pSV3gptH to the UV-sensitive, incision-defective Chinese hamster ovary (CHO) mutant 27-1. This mutant belongs to complementation group 3 of repair-deficient rodent mutants. After selection of UV-resistant primary and secondary 27-1 transformants, human sequences associated with the induced UV resistance were rescued in cosmids from the DNA of a secondary transformant by using a linked dominant marker copy and human repetitive DNA as probes. From coinheritance analysis of the ERCC-3 region in independent transformants, we deduce that the gene has a size of 35 to 45 kilobases, of which one essential segment has so far been refractory to cloning. Conserved unique human sequences hybridizing to a 3.0-kilobase mRNA were used to isolate apparently full-length cDNA clones. Upon transfection to 27-1 cells, the ERCC-3 cDNA, inserted in a mammalian expression vector, induced specific and (virtually) complete correction of the UV sensitivity and unscheduled DNA synthesis of mutants of complementation group 3 with very high efficiency. Mutant 27-1 is, unlike other mutants of complementation group 3, also very sensitive toward small alkylating agents. This unique property of the mutant is not corrected by introduction of the ERCC-3 cDNA, indicating that it may be caused by an independent second mutation in another repair function. By hybridization to DNA of a human x rodent hybrid cell panel, the ERCC-3 gene was assigned to chromosome 2, in agreement with data based on cell fusion (L. H. Thompson, A. V. Carrano, K. Sato, E. P. Salazar, B. F. White, S. A. Stewart, J. L. Minkler, and M. J. Siciliano, Somat. Cell. Mol. Genet. 13:539-551, 1987).

Download Full-text