High Free-Energy Barrier of 1D Diffusion Along DNA by Architectural DNA-Binding Proteins

Kamagata, Kiyoto; Mano, Eriko; Ouchi, Kota; Kanbayashi, Saori; Johnson, Reid C.

doi:10.1016/j.jmb.2018.01.001

Cited by 34 publications

(80 citation statements)

References 96 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Native or proteins containing one or two cysteines for labeling were expressed and purified without tags as described previously ( 12 ). Two versions of Nhp6A for labeling were employed: Nhp6A-A93C contains a cysteine substituted for the C-terminal alanine residue, and Nhp6A-2cys contains cysteines introduced at residue 2 and the C-terminal end.…”

Section: Methodsmentioning

confidence: 99%

“…The HU β subunit, containing a cysteine added to its C-terminal end, and HU α subunit were co-expressed in E. coli to form HU heterodimers. The proteins were labeled by Atto488 (ATTO-TEC) using maleimido chemistry as reported previously ( 12 ).…”

Section: Methodsmentioning

confidence: 99%

“…Previously, we investigated the behavior of these three ADBPs on naked DNA using single-molecule fluorescence microscopy ( 12 ). We found that these ADBPs support 1D diffusion along DNA while maintaining continuous DNA contact, although Fis most often associates with DNA in a stationary mode.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

The HMGB chromatin protein Nhp6A can bypass obstacles when traveling on DNA

Kamagata

Ouchi

Tan

et al. 2020

Nucleic Acids Research

Self Cite

View full text Add to dashboard Cite

DNA binding proteins rapidly locate their specific DNA targets through a combination of 3D and 1D diffusion mechanisms, with the 1D search involving bidirectional sliding along DNA. However, even in nucleosome-free regions, chromosomes are highly decorated with associated proteins that may block sliding. Here we investigate the ability of the abundant chromatin-associated HMGB protein Nhp6A from Saccharomyces cerevisiae to travel along DNA in the presence of other architectural DNA binding proteins using single-molecule fluorescence microscopy. We observed that 1D diffusion by Nhp6A molecules is retarded by increasing densities of the bacterial proteins Fis and HU and by Nhp6A, indicating these structurally diverse proteins impede Nhp6A mobility on DNA. However, the average travel distances were larger than the average distances between neighboring proteins, implying Nhp6A is able to bypass each of these obstacles. Together with molecular dynamics simulations, our analyses suggest two binding modes: mobile molecules that can bypass barriers as they seek out DNA targets, and near stationary molecules that are associated with neighboring proteins or preferred DNA structures. The ability of mobile Nhp6A molecules to bypass different obstacles on DNA suggests they do not block 1D searches by other DNA binding proteins.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

The HMGB chromatin protein Nhp6A can bypass obstacles when traveling on DNA

Kamagata

Ouchi

Tan

et al. 2020

Nucleic Acids Research

Self Cite

View full text Add to dashboard Cite

show abstract

“…Since many DNA-binding proteins exist in the cell and have different structures and functions, it is interesting how such DNA-binding proteins solve the target search problem. For example, if certain DNA-binding proteins differ in structure and function, their target search mechanisms may each differ as well [101][102][103][104][105]. In the near future, the target search mechanism of various DNA-binding proteins will be revealed by single-molecule fluorescence microscopy.…”

Section: Summary and Future Perspectivesmentioning

confidence: 99%

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

Kamagata

Itoh

Subekti

2020

IJMS

Self Cite

View full text Add to dashboard Cite

Interactions between DNA and DNA-binding proteins play an important role in many essential cellular processes. A key function of the DNA-binding protein p53 is to search for and bind to target sites incorporated in genomic DNA, which triggers transcriptional regulation. How do p53 molecules achieve “rapid” and “accurate” target search in living cells? The search dynamics of p53 were expected to include 3D diffusion in solution, 1D diffusion along DNA, and intersegmental transfer between two different DNA strands. Single-molecule fluorescence microscopy enabled the tracking of p53 molecules on DNA and the characterization of these dynamics quantitatively. Recent intensive single-molecule studies of p53 succeeded in revealing each of these search dynamics. Here, we review these studies and discuss the target search mechanisms of p53.

show abstract

“…The disordered linker enables switching between the two sliding modes 22 . p53 slides along DNA rotationally following the DNA groove 15,25 . Target recognition by the sliding p53 is quite low and can be regulated by single mutations 8 .…”

mentioning

confidence: 99%

Transient binding and jumping dynamics of p53 along DNA revealed by sub-millisecond resolved single-molecule fluorescence tracking

Subekti

Murata

Itoh

et al. 2020

Sci Rep

Self Cite

View full text Add to dashboard Cite

Characterization of the target search dynamics of DNA-binding proteins along DNA has been hampered by the time resolution of a standard single-molecule fluorescence microscopy. Here, we achieved the time resolution of 0.5 ms in the fluorescence microscopy measurements by optimizing the fluorescence excitation based on critical angle illumination and by utilizing the time delay integration mode of the electron-multiplying charge coupled device. We characterized the target search dynamics of the tumor suppressor p53 along nonspecific DNA at physiological salt concentrations. We identified a short-lived encounter intermediate before the formation of the longlived p53-DNA complex. Both the jumps and the one-dimensional diffusion of p53 along DNA were accelerated at higher salt concentrations, suggesting the rotation-uncoupled movement of p53 along DNA grooves and conformational changes in the p53/DNA complex. This method can be used to clarify the unresolved dynamics of DNA-binding proteins previously hidden by time averaging. Sequence-specific DNA-binding proteins bind to their respective target DNAs accurately and quickly and participate in the control and maintenance of cellular functions. To facilitate the search for target sequences among enormous lengths of DNA, these proteins utilize a strategy called facilitated diffusion, which involves a combination of various dynamics, such as one-dimensional (1D) diffusion and jumping along nontarget DNA, three dimensional (3D) diffusion between two separated sites of DNA, and intersegmental transfer at the contact points of two DNA strands 1-6. Inhibiting the search for and binding to the target DNA by these proteins will lead to insufficient control of cell functions, which could result in disease 7,8. The prevailing method for the characterization of the target search dynamics of sequence-specific DNAbinding proteins is single-molecule fluorescence microscopy in combination with tethering and stretching of DNA on the surface of flow cells 9-15. Typically, DNA-binding proteins labeled with a fluorescent dye or a quantum dot are introduced into the flow cell, allowed to interact with the stretched DNA, and excited selectively by highly inclined and laminated optical sheet (HILO) illumination or by total internal reflection fluorescence (TIRF). Fluorescent spots from single proteins are sequentially tracked using imaging detectors, such as electron-multiplying charge coupled devices (EM-CCDs), enabling the characterization of their target search dynamics. The time resolution of the method is determined by two factors. First, detection of fluorescent molecules at a spatial resolution of ~ 40 nm requires the collection of at least 50 photons, which in turn requires a certain period of data accumulation and sets the time resolution. Second, the frame rate of the imaging detectors (typically 10-50/s) also sets the time resolution. The shortest reported time resolution for localization of DNA-binding proteins along stretched DNA is 8 ms at a spatial resolution of 22-42 nm 16. ...

show abstract

High Free-Energy Barrier of 1D Diffusion Along DNA by Architectural DNA-Binding Proteins

Cited by 34 publications

References 96 publications

The HMGB chromatin protein Nhp6A can bypass obstacles when traveling on DNA

The HMGB chromatin protein Nhp6A can bypass obstacles when traveling on DNA

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

Transient binding and jumping dynamics of p53 along DNA revealed by sub-millisecond resolved single-molecule fluorescence tracking

Contact Info

Product

Resources

About