Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Resolving the subtle details of human DNA alkyltransferase lesion search and repair mechanism by single-molecule studies.

ABSTRACT: SignificanceWe directly visualize DNA translocation and lesion recognition by the O⁶-alkylguanine DNA alkyltransferase (AGT). Our data show bidirectional movement of AGT monomers and clusters on undamaged DNA that depended on Zn²⁺ occupancy of AGT. A role of cooperative AGT clusters in enhancing lesion search efficiencies by AGT has previously been proposed. Surprisingly, our data show no enhancement of DNA translocation speed by AGT cluster formation, suggesting that AGT clusters may serve a different role in AGT function. Our data support preferential cluster formation by AGT at alkyl lesions, suggesting a role of these clusters in stabilizing lesion-bound complexes. From our data, we derive a new model for the lesion search and repair mechanism of AGT.

SUBMITTER: Kono S

PROVIDER: S-EPMC8931253 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Resolving the subtle details of human DNA alkyltransferase lesion search and repair mechanism by single-molecule studies.

Kono Sarah S van den Berg Aafke A Simonetta Marco M Mukhortava Ann A Garman Elspeth F EF Tessmer Ingrid I

Proceedings of the National Academy of Sciences of the United States of America 20220308 11

SignificanceWe directly visualize DNA translocation and lesion recognition by the O<sup>6</sup>-alkylguanine DNA alkyltransferase (AGT). Our data show bidirectional movement of AGT monomers and clusters on undamaged DNA that depended on Zn<sup>2+</sup> occupancy of AGT. A role of cooperative AGT clusters in enhancing lesion search efficiencies by AGT has previously been proposed. Surprisingly, our data show no enhancement of DNA translocation speed by AGT cluster formation, suggesting that AGT c ...[more]

PMID: 35259021

Similar Datasets

Mechanism of homology search expansion during recombinational DNA repair

Project description:Homology search is a central step of DNA double-strand break (DSB) repair by homologous recombination. How it operates in cells remains elusive. Here we developed a Hi-C-based methodology to map single-stranded DNA (ssDNA) contacts genome-wide in S. cerevisiae, which revealed two main homology search phases. Initial search conducted by short Rad51-ssDNA nucleoprotein filaments (NPFs) is confined in cis by cohesin-mediated chromatin loop folding. Progressive growth of stiff NPFs enables exploration of distant genomic sites. Long-range resection drives this transition from local to genome-wide search by increasing the probability of assembly of extensive NPFs. DSB end-tethering promotes coordinated homology search by opposite NPFs. Finally, an autonomous genetic element on chromosome III engages the NPF, which stimulates homology search in its vicinity. This work reveals the mechanism of the progressive expansion of homology search orchestrated by chromatin organizers, long-range resection, end-tethering, specialized genetic elements, and that exploits the stiff NPF structure conferred by Rad51 oligomerization.

2024-07-29 | GSE272969 | GEO

Lesion search and recognition by thymine DNA glycosylase revealed by single molecule imaging.

Project description:The ability of DNA glycosylases to rapidly and efficiently detect lesions among a vast excess of nondamaged DNA bases is vitally important in base excision repair (BER). Here, we use single molecule imaging by atomic force microscopy (AFM) supported by a 2-aminopurine fluorescence base flipping assay to study damage search by human thymine DNA glycosylase (hTDG), which initiates BER of mutagenic and cytotoxic G:T and G:U mispairs in DNA. Our data reveal an equilibrium between two conformational states of hTDG-DNA complexes, assigned as search complex (SC) and interrogation complex (IC), both at target lesions and undamaged DNA sites. Notably, for both hTDG and a second glycosylase, hOGG1, which recognizes structurally different 8-oxoguanine lesions, the conformation of the DNA in the SC mirrors innate structural properties of their respective target sites. In the IC, the DNA is sharply bent, as seen in crystal structures of hTDG lesion recognition complexes, which likely supports the base flipping required for lesion identification. Our results support a potentially general concept of sculpting of glycosylases to their targets, allowing them to exploit the energetic cost of DNA bending for initial lesion sensing, coupled with continuous (extrahelical) base interrogation during lesion search by DNA glycosylases.

| S-EPMC4357730 | biostudies-literature

Single-molecule imaging reveals target-search mechanisms during DNA mismatch repair.

Project description:The ability of proteins to locate specific targets among a vast excess of nonspecific DNA is a fundamental theme in biology. Basic principles governing these search mechanisms remain poorly understood, and no study has provided direct visualization of single proteins searching for and engaging target sites. Here we use the postreplicative mismatch repair proteins MutS? and MutL? as model systems for understanding diffusion-based target searches. Using single-molecule microscopy, we directly visualize MutS? as it searches for DNA lesions, MutL? as it searches for lesion-bound MutS?, and the MutS?/MutL? complex as it scans the flanking DNA. We also show that MutL? undergoes intersite transfer between juxtaposed DNA segments while searching for lesion-bound MutS?, but this activity is suppressed upon association with MutS?, ensuring that MutS/MutL remains associated with the damage-bearing strand while scanning the flanking DNA. Our findings highlight a hierarchy of lesion- and ATP-dependent transitions involving both MutS? and MutL?, and help establish how different modes of diffusion can be used during recognition and repair of damaged DNA.

| S-EPMC3494904 | biostudies-literature

Single molecule studies of DNA mismatch repair.

Project description:DNA mismatch repair, which involves is a widely conserved set of proteins, is essential to limit genetic drift in all organisms. The same system of proteins plays key roles in many cancer related cellular transactions in humans. Although the basic process has been reconstituted in vitro using purified components, many fundamental aspects of DNA mismatch repair remain hidden due in part to the complexity and transient nature of the interactions between the mismatch repair proteins and DNA substrates. Single molecule methods offer the capability to uncover these transient but complex interactions and allow novel insights into mechanisms that underlie DNA mismatch repair. In this review, we discuss applications of single molecule methodology including electron microscopy, atomic force microscopy, particle tracking, FRET, and optical trapping to studies of DNA mismatch repair. These studies have led to formulation of mechanistic models of how proteins identify single base mismatches in the vast background of matched DNA and signal for their repair.

| S-EPMC4310750 | biostudies-literature

Alkyltransferase-like proteins: molecular switches between DNA repair pathways.

Project description:Alkyltransferase-like proteins (ATLs) play a role in the protection of cells from the biological effects of DNA alkylation damage. Although ATLs share functional motifs with the DNA repair protein and cancer chemotherapy target O?-alkylguanine-DNA alkyltransferase, they lack the reactive cysteine residue required for alkyltransferase activity, so its mechanism for cell protection was previously unknown. Here we review recent advances in unraveling the enigmatic cellular protection provided by ATLs against the deleterious effects of DNA alkylation damage. We discuss exciting new evidence that ATLs aid in the repair of DNA O?-alkylguanine lesions through a novel repair cross-talk between DNA-alkylation base damage responses and the DNA nucleotide excision repair pathway.

| S-EPMC3088651 | biostudies-literature

Resolving metal-molecule interfaces at single-molecule junctions.

Project description:Electronic and structural detail at the electrode-molecule interface have a significant influence on charge transport across molecular junctions. Despite the decisive role of the metal-molecule interface, a complete electronic and structural characterization of the interface remains a challenge. This is in no small part due to current experimental limitations. Here, we present a comprehensive approach to obtain a detailed description of the metal-molecule interface in single-molecule junctions, based on current-voltage (I-V) measurements. Contrary to conventional conductance studies, this I-V approach provides a correlated statistical description of both, the degree of electronic coupling across the metal-molecule interface, and the energy alignment between the conduction orbital and the Fermi level of the electrode. This exhaustive statistical approach was employed to study single-molecule junctions of 1,4-benzenediamine (BDA), 1,4-butanediamine (C4DA), and 1,4-benzenedithiol (BDT). A single interfacial configuration was observed for both BDA and C4DA junctions, while three different interfacial arrangements were resolved for BDT. This multiplicity is due to different molecular adsorption sites on the Au surface namely on-top, hollow, and bridge. Furthermore, C4DA junctions present a fluctuating I-V curve arising from the greater conformational freedom of the saturated alkyl chain, in sharp contrast with the rigid aromatic backbone of both BDA and BDT.

| S-EPMC4879565 | biostudies-literature

Mechanism of lesion verification by the human XPD helicase in nucleotide excision repair.

Project description:In nucleotide excision repair (NER), the xeroderma pigmentosum D helicase (XPD) scans DNA searching for bulky lesions, stalls when encountering such damage to verify its presence, and allows repair to proceed. Structural studies have shown XPD bound to its single-stranded DNA substrate, but molecular and dynamic characterization of how XPD translocates on undamaged DNA and how it stalls to verify lesions remains poorly understood. Here, we have performed extensive all-atom MD simulations of human XPD bound to undamaged and damaged ssDNA, containing a mutagenic pyrimidine (6-4) pyrimidone UV photoproduct (6-4PP), near the XPD pore entrance. We characterize how XPD responds to the presence of the DNA lesion, delineating the atomistic-scale mechanism that it utilizes to discriminate between damaged and undamaged nucleotides. We identify key amino acid residues, including FeS residues R112, R196, H135, K128, Arch residues E377 and R380, and ATPase lobe 1 residues 215-221, that are involved in damage verification and show how movements of Arch and ATPase lobe 1 domains relative to the FeS domain modulate these interactions. These structural and dynamic molecular depictions of XPD helicase activity with unmodified DNA and its inhibition by the lesion elucidate how the lesion is verified by inducing XPD stalling.

| S-EPMC9262607 | biostudies-literature

Mechanism of homology search expansion during recombinational DNA repair

Project description:Mechanism of homology search expansion during recombinational DNA repair

| PRJNA1139728 | ENA

Advances in Structural and Single-Molecule Methods for Investigating DNA Lesion Bypass and Repair Polymerases.

Project description:Innovative advances in X-ray crystallography and single-molecule biophysics have yielded unprecedented insight into the mechanisms of DNA lesion bypass and damage repair. Time-dependent X-ray crystallography has been successfully applied to view the bypass of 8-oxo-7,8-dihydro-2'-deoxyguanine (8-oxoG), a major oxidative DNA lesion, and the incorporation of the triphosphate form, 8-oxo-dGTP, catalyzed by human DNA polymerase β. Significant findings of these studies are highlighted here, and their contributions to the current mechanistic understanding of mutagenic translesion DNA synthesis (TLS) and base excision repair are discussed. In addition, single-molecule Förster resonance energy transfer (smFRET) techniques have recently been adapted to investigate nucleotide binding and incorporation opposite undamaged dG and 8-oxoG by Sulfolobus solfataricus DNA polymerase IV (Dpo4), a model Y-family DNA polymerase. The mechanistic response of Dpo4 to a DNA lesion and the complex smFRET technique are described here. In this perspective, we also describe how time-dependent X-ray crystallography and smFRET can be used to achieve the spatial and temporal resolutions necessary to answer some of the mechanistic questions that remain in the fields of TLS and DNA damage repair.

| S-EPMC6634979 | biostudies-literature

Lesion specificity in the base excision repair enzyme hNeil1: modeling and dynamics studies.

Project description:Base excision repair (BER) is the major pathway employed to excise oxidized DNA lesions. Human Neil1, a versatile glycosylase in the BER pathway, repairs a diverse array of oxidative lesions; however, the most prevalent, 8-oxo-7,8-dihydroguanine (8-oxoG), is only weakly excised. The structural origin of hNeil1's ability to repair a variety of lesions but not 8-oxoG is a model system for connecting enzyme structure and lesion-recognition specificity. To elucidate structural properties determining hNeil1's substrate specificities, we have investigated it in complex with two pairs of representative well-repaired substrates: the R- and S-spiroiminodihydantoin (Sp) stereoisomers, nonplanar further oxidation products of guanine, and the 5R,6S- and 5S,6R-thymine glycol (Tg) stereoisomers, the most prevalent oxidative lesions of thymine. We also investigate the poorly repaired 8-oxoG. We employed molecular modeling and 10 ns molecular dynamics (MD) simulations. The results of our investigations provide structural explanations for the ability of hNeil1 to excise a variety of oxidative lesions: they possess common chemical features, namely, a pyrimidine-like ring and shared hydrogen bond donor-acceptor properties, which allow the lesions to fit well in the binding pocket, which is somewhat flexible. However, the planar 8-oxoG is not as well accommodated in the shallow and comparatively cramped recognition pocket; it has fewer hydrogen bonding interactions with the enzyme and a solvent exposed six-membered ring, consistent with its poor repair susceptibility by this enzyme.

| S-EPMC2527061 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data