scholarly journals Sequence specificity, energetics and mechanism of mismatch recognition by DNA damage sensing protein Rad4/XPC

2020 ◽  
Vol 48 (5) ◽  
pp. 2246-2257 ◽  
Author(s):  
Abhinandan Panigrahi ◽  
Hemanth Vemuri ◽  
Madhur Aggarwal ◽  
Kartheek Pitta ◽  
Marimuthu Krishnan
Keyword(s):  
Molecular Basis ◽  
High Specificity ◽  
Umbrella Sampling ◽  
Dna Lesions ◽  
Sequence Specificity ◽  
Dna Complexes ◽  
Mismatched Dna ◽  
Damage Sensing ◽  
Molecular Events ◽  
Mismatch Recognition

Abstract The ultraviolet (UV) radiation-induced DNA lesions play a causal role in many prevalent genetic skin-related diseases and cancers. The damage sensing protein Rad4/XPC specifically recognizes and repairs these lesions with high fidelity and safeguards genome integrity. Despite considerable progress, the mechanistic details of the mode of action of Rad4/XPC in damage recognition remain obscure. The present study investigates the mechanism, energetics, dynamics, and the molecular basis for the sequence specificity of mismatch recognition by Rad4/XPC. We dissect the following three key molecular events that occur as Rad4/XPC tries to recognize and bind to DNA lesions/mismatches: (a) the association of Rad4/XPC with the damaged/mismatched DNA, (b) the insertion of a lesion-sensing β-hairpin of Rad4/XPC into the damage/mismatch site and (c) the flipping of a pair of nucleotide bases at the damage/mismatch site. Using suitable reaction coordinates, the free energy surfaces for these events are determined using molecular dynamics (MD) and umbrella sampling simulations on three mismatched (CCC/CCC, TTT/TTT and TAT/TAT mismatches) Rad4-DNA complexes. The study identifies the key determinants of the sequence-dependent specificity of Rad4 for the mismatches and explores the ramifications of specificity in the aforementioned events. The results unravel the molecular basis for the high specificity of Rad4 towards CCC/CCC mismatch and lower specificity for the TAT/TAT mismatch. A strong correlation between the depth of β-hairpin insertion into the DNA duplex and the degree of coupling between the hairpin insertion and the flipping of bases is also observed. The interplay of the conformational flexibility of mismatched bases, the depth of β-hairpin insertion, Rad4-DNA association energetics and the Rad4 specificity explored here complement recent experimental FRET studies on Rad4-DNA complexes.

scholarly journals Excision of Oxidatively Generated Guanine Lesions by Competitive DNA Repair Pathways

2021 ◽  
Vol 22 (5) ◽  
pp. 2698
Author(s):  
Vladimir Shafirovich ◽  
Nicholas E. Geacintov
Keyword(s):  
Excision Repair ◽  
Dna Lesions ◽  
Dna Templates ◽  
Circular Dna ◽  
Damage Sensing ◽  
Cell Extracts ◽  
Dna Base ◽  
Nucleotide Excision ◽  
Repair Pathways ◽  
Cellular Dna

The base and nucleotide excision repair pathways (BER and NER, respectively) are two major mechanisms that remove DNA lesions formed by the reactions of genotoxic intermediates with cellular DNA. It is generally believed that small non-bulky oxidatively generated DNA base modifications are removed by BER pathways, whereas DNA helix-distorting bulky lesions derived from the attack of chemical carcinogens or UV irradiation are repaired by the NER machinery. However, existing and growing experimental evidence indicates that oxidatively generated DNA lesions can be repaired by competitive BER and NER pathways in human cell extracts and intact human cells. Here, we focus on the interplay and competition of BER and NER pathways in excising oxidatively generated guanine lesions site-specifically positioned in plasmid DNA templates constructed by a gapped-vector technology. These experiments demonstrate a significant enhancement of the NER yields in covalently closed circular DNA plasmids (relative to the same, but linearized form of the same plasmid) harboring certain oxidatively generated guanine lesions. The interplay between the BER and NER pathways that remove oxidatively generated guanine lesions are reviewed and discussed in terms of competitive binding of the BER proteins and the DNA damage-sensing NER factor XPC-RAD23B to these lesions.

scholarly journals Epigenetic events in medulloblastoma development

2005 ◽  
Vol 19 (5) ◽  
pp. 1-13 ◽  
Author(s):  
Janet C. Lindsey ◽  
Jennifer A. Anderton ◽  
Meryl E. Lusher ◽  
Steven C. Clifford
Keyword(s):  
Gene Expression ◽  
Tumor Suppressor Genes ◽  
Molecular Basis ◽  
Gene Disruption ◽  
Functional Role ◽  
Promoter Hypermethylation ◽  
Therapeutic Approaches ◽  
Primary Tumors ◽  
Molecular Events ◽  
Epigenetic Events

Over the last decade, the analysis of genetic defects in primary tumors has been central to the identification of molecular events and biological pathways involved in the pathogenesis of medulloblastoma, the most common malignant brain tumor of childhood. Despite this, understanding of the molecular basis of the majority of cases remains poor. In recent years, the emerging field of epigenetics, which describes heritable alterations in gene expression that occur in the absence of DNA sequence changes, has forced a revision of the understanding of the mechanisms of gene disruption in cancer. Accumulating evidence indicates a significant involvement for epigenetic events in medulloblastoma development. Recent studies have identified a series of candidate tumor suppressor genes (for example, RASSF1A, CASP8, and HIC1) that are each specifically epigenetically inactivated in a large proportion (> 30%) of medulloblastomas by promoter hypermethylation, leading to the silencing of their gene expression. These findings shed new light on medulloblastoma and offer great potential for an improved understanding of its molecular pathology. The authors review the current understanding of epigenetic events in cancer and their contribution to medulloblastoma development. Their nature, origins, and functional role(s) in tumorigenesis are considered, and the authors assess the potential utility of these events as a basis for novel diagnostic and therapeutic approaches.

Inhibition of transcription factor assembly and structural stability on mitoxantrone binding with DNA

2010 ◽  
Vol 30 (5) ◽  
pp. 331-340 ◽  
Author(s):  
Shahper N. Khan ◽  
Mohd Danishuddin ◽  
Asad U. Khan
Keyword(s):  
Transcription Factor ◽  
Binding Mode ◽  
Cd Spectroscopy ◽  
Sequence Specificity ◽  
Mobility Shift ◽  
Dna Complexes ◽  
Naked Dna ◽  
Plasmid Nicking Assay ◽  
Modelling Studies ◽  
Mobility Shift Assay

MTX (mitoxantrone) is perhaps the most promising drug used in the treatment of various malignancies. Comprehensive literature on the therapeutics has indicated it to be the least toxic in its class, although its mechanism of action is still not well defined. In the present study, we have evaluated the associated binding interactions of MTX with naked DNA. The mechanism of MTX binding with DNA was elucidated by steady-state fluorescence and a static-type quenching mechanism is suggested for this interaction. Thermodynamic parameters from van 't Hoff plots showed that the interaction of these drugs with DNA is an entropically driven phenomenon. The binding mode was expounded by attenuance measurements and competitive binding of a known intercalator. Sequence specificity of these drug–DNA complexes was analysed by FTIR (Fourier-transform infrared) spectroscopy and molecular modelling studies. CD spectroscopy and the plasmid nicking assay showed that the binding of this drug with DNA results in structural and conformational perturbations. EMSA (electrophoretic mobility-shift assay) results showed that these drug–DNA complexes prevent the binding of octamer TF (transcription factor) to DNA. In summary, the study implicates MTX-induced conformational instability and transcription inhibition on DNA binding.

scholarly journals Dynamics of MutS–Mismatched DNA Complexes Are Predictive of Their Repair Phenotypes

Biochemistry ◽  
2014 ◽  
Vol 53 (12) ◽  
pp. 2043-2052 ◽  
Author(s):  
Vanessa C. DeRocco ◽  
Lauryn E. Sass ◽  
Ruoyi Qiu ◽  
Keith R. Weninger ◽  
Dorothy A. Erie
Keyword(s):  
Dna Complexes ◽  
Mismatched Dna

scholarly journals Dynamic human MutSα–MutLα complexes compact mismatched DNA

2020 ◽  
Vol 117 (28) ◽  
pp. 16302-16312 ◽  
Author(s):  
Kira C. Bradford ◽  
Hunter Wilkins ◽  
Pengyu Hao ◽  
Zimeng M. Li ◽  
Bangchen Wang ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Proliferating Cell Nuclear Antigen ◽  
Nuclear Antigen ◽  
Dna Complexes ◽  
Force Microscopy ◽  
Dna Mismatch ◽  
Mismatched Dna ◽  
Atomic Force ◽  
Higher Eukaryotes ◽  
Proliferating Cell

DNA mismatch repair (MMR) corrects errors that occur during DNA replication. In humans, mutations in the proteins MutSα and MutLα that initiate MMR cause Lynch syndrome, the most common hereditary cancer. MutSα surveilles the DNA, and upon recognition of a replication error it undergoes adenosine triphosphate-dependent conformational changes and recruits MutLα. Subsequently, proliferating cell nuclear antigen (PCNA) activates MutLα to nick the error-containing strand to allow excision and resynthesis. The structure–function properties of these obligate MutSα–MutLα complexes remain mostly unexplored in higher eukaryotes, and models are predominately based on studies of prokaryotic proteins. Here, we utilize atomic force microscopy (AFM) coupled with other methods to reveal time- and concentration-dependent stoichiometries and conformations of assembling human MutSα–MutLα–DNA complexes. We find that they assemble into multimeric complexes comprising three to eight proteins around a mismatch on DNA. On the timescale of a few minutes, these complexes rearrange, folding and compacting the DNA. These observations contrast with dominant models of MMR initiation that envision diffusive MutS–MutL complexes that move away from the mismatch. Our results suggest MutSα localizes MutLα near the mismatch and promotes DNA configurations that could enhance MMR efficiency by facilitating MutLα nicking the DNA at multiple sites around the mismatch. In addition, such complexes may also protect the mismatch region from nucleosome reassembly until repair occurs, and they could potentially remodel adjacent nucleosomes.

scholarly journals The molecular basis for recognition of 5′-NNNCC-3′ PAM and its methylation state by Acidothermus cellulolyticus Cas9

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Anuska Das ◽  
Travis H. Hand ◽  
Chardasia L. Smith ◽  
Ethan Wickline ◽  
Michael Zawrotny ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Methylation Status ◽  
Extreme Environments ◽  
High Sensitivity ◽  
High Specificity ◽  
Acidothermus Cellulolyticus ◽  
Guide Rna ◽  
Protospacer Adjacent Motif ◽  
Bond Network ◽  
Rna And Dna

AbstractAcidothermus cellulolyticus CRISPR-Cas9 (AceCas9) is a thermophilic Type II-C enzyme that has potential genome editing applications in extreme environments. It cleaves DNA with a 5′-NNNCC-3′ Protospacer Adjacent Motif (PAM) and is sensitive to its methylation status. To understand the molecular basis for the high specificity of AceCas9 for its PAM, we determined two crystal structures of AceCas9 lacking its HNH domain (AceCas9-ΔHNH) bound with a single guide RNA and DNA substrates, one with the correct and the other with an incorrect PAM. Three residues, Glu1044, Arg1088, Arg1091, form an intricate hydrogen bond network with the first cytosine and the two opposing guanine nucleotides to confer specificity. Methylation of the first but not the second cytosine base abolishes AceCas9 activity, consistent with the observed PAM recognition pattern. The high sensitivity of AceCas9 to the modified cytosine makes it a potential device for detecting epigenomic changes in genomes.

Single-molecule micromanipulation studies of DNA and architectural proteins

2008 ◽  
Vol 36 (4) ◽  
pp. 732-737 ◽  
Author(s):  
Remus Th. Dame
Keyword(s):  
Dna Binding ◽  
Single Molecule ◽  
Genomic Dna ◽  
Dna Binding Proteins ◽  
Binding Kinetics ◽  
Sequence Specificity ◽  
Effective Volume ◽  
Dna Complexes ◽  
Ensemble Techniques ◽  
Architectural Proteins

Architectural proteins play a key role in the folding, organization and compaction of genomic DNA in all organisms. By bending, bridging or wrapping DNA, these proteins ensure that its effective volume is reduced sufficiently to fit inside the cell or a dedicated cellular organelle, the nucleus (in bacteria/archaea and in eukaryotes respectively). In addition, the properties of many of these proteins permit them to play specific roles as architectural cofactors in a large variety of DNA transactions. However, as architectural proteins often bind DNA with low sequence specificity and affinity, it is hard to investigate their interaction using biochemical ensemble techniques. Single-molecule micromanipulation approaches that probe the properties of DNA-binding proteins by pulling on individual protein–DNA complexes have, in this respect, proved to be a very powerful alternative. Besides revealing architectural properties, these approaches can also reveal unique parameters not accessible to biochemical approaches, such as the binding kinetics and unbinding forces of individual proteins.

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