How p53 Molecules Solve the Target DNA Search Problem: A Review

Kamagata, Kiyoto; Itoh, Yuji; Subekti, Dwiky Rendra Graha

doi:10.3390/ijms21031031

Cited by 23 publications

(20 citation statements)

References 104 publications

(122 reference statements)

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“…Comprehensive mutagenesis analysis supports the correlation between the structured domains of p53 and its function [7]. Since p53 possesses common properties frequently observed in DNA-binding proteins, including oligomerization, disordered regions, and multiple DNA-binding domains [8], it is used as a model protein in the target search study described below [9][10][11].…”

Section: Introductionmentioning

confidence: 74%

A Study of p53 Action on DNA at the Single Molecule Level

Kamagata¹

2022

P53 - A Guardian of the Genome and Beyond

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The transcription factor p53 searches for and binds to target sequences within long genomic DNA, to regulate downstream gene expression. p53 possesses multiple disordered and DNA-binding domains, which are frequently observed in DNA-binding proteins. Owing to these properties, p53 is used as a model protein for target search studies. It counters cell stress by utilizing a facilitated diffusion mechanism that combines 3D diffusion in solution, 1D sliding along DNA, hopping/jumping along DNA, and intersegmental transfer between two DNAs. Single-molecule fluorescence microscopy has been used to characterize individual motions of p53 in detail. In addition, a biophysical study has revealed that p53 forms liquid-like droplets involving the functional switch. In this chapter, the target search and regulation of p53 are discussed in terms of dynamic properties.

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Section: Introductionmentioning

confidence: 74%

A Study of p53 Action on DNA at the Single Molecule Level

Kamagata¹

2022

P53 - A Guardian of the Genome and Beyond

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“…In the 21st century, single-molecule fluorescence tracking of proteins bound to DNA has gained widespread popularity in the biophysical field [ 8 , 10 , 14 , 19 – 21 , 31 ]. The tacking for thousands of proteins molecules bound to DNA allows for quantitative analysis of sliding and dissociation.…”

Section: Experimental Applicationsmentioning

confidence: 99%

“…This work was remarkable in that these hypothetical concepts were postulated when only a very limited number of experimental methods and no crystal structures of protein-DNA complexes were available. Now, sliding of proteins on DNA is a well-established fact, which has been directly observed for many DNA-binding proteins in vitro and even in vivo by single-molecule methods [ 8 , 10 , 14 , 19 – 21 , 31 ]. Other methods such as nuclear magnetic resonance (NMR) spectroscopy [ 13 , 32 ], stopped-flow fluorescence [ 33 – 35 ], and elaborate biochemical approaches [ 36 – 42 ] also provide rich and quantitative information about how proteins locate their targets on DNA.…”

Section: Introductionmentioning

confidence: 99%

Discrete-state stochastic kinetic models for target DNA search by proteins: Theory and experimental applications

Iwahara

Kolomeisky

2021

Biophysical Chemistry

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To perform their functions, transcription factors and DNA-repair/modifying enzymes randomly search DNA in order to locate their specific targets on DNA. Discrete-state stochastic kinetic models have been developed to explain how the efficiency of the search process is influenced by the molecular properties of proteins and DNA as well as by other factors such as molecular crowding. These theoretical models not only offer explanations on the relation of microscopic processes to macroscopic behavior of proteins, but also facilitate the analysis and interpretation of experimental data. In this review article, we provide an overview on discrete-state stochastic kinetic models and explain how these models can be applied to experimental investigations using stopped-flow, single-molecule, nuclear magnetic resonance (NMR), and other biophysical and biochemical methods.

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“…The target search by 3D diffusion was unique to Cas9. In contrast, 1D diffusion (sliding) along DNA, as well as 3D diffusion in solution, called a facilitated diffusion mechanism, are utilized by many DNA-binding proteins in these target searches 36 – 38 . If Cas9 can slide along the DNA, the target search time would be reduced dramatically, thus promoting the double-strand breaks and following a subsequent reaction like non-homologous end joining (NHEJ) or homology-directed repair (HDR).…”

Section: Introductionmentioning

confidence: 99%

Engineering of the genome editing protein Cas9 to slide along DNA

Banerjee

Takahashi

Subekti

et al. 2021

Sci Rep

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The genome editing protein Cas9 faces engineering challenges in improving off–target DNA cleavage and low editing efficiency. In this study, we aimed to engineer Cas9 to be able to slide along DNA, which might facilitate genome editing and reduce off-target cleavage. We used two approaches to achieve this: reducing the sliding friction along DNA by removing the interactions of Cas9 residues with DNA and facilitating sliding by introducing the sliding-promoting tail of Nhp6A. Seven engineered mutants of Cas9 were prepared, and their performance was tested using single-molecule fluorescence microscopy. Comparison of the mutations enabled the identification of key residues of Cas9 to enhance the sliding along DNA in the presence and absence of single guide RNA (sgRNA). The attachment of the tail to Cas9 mutants enhanced sliding along DNA, particularly in the presence of sgRNA. Together, using the proposed approaches, the sliding ability of Cas9 was improved up to eightfold in the presence of sgRNA. A sliding model of Cas9 and its engineering action are discussed herein.

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How p53 Molecules Solve the Target DNA Search Problem: A Review

Cited by 23 publications

References 104 publications

A Study of p53 Action on DNA at the Single Molecule Level

A Study of p53 Action on DNA at the Single Molecule Level

Discrete-state stochastic kinetic models for target DNA search by proteins: Theory and experimental applications

Engineering of the genome editing protein Cas9 to slide along DNA

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