Protein Sliding along DNA: Dynamics and Structural Characterization

Efficient search of DNA by proteins is fundamental to the control of cellular regulatory processes. It is currently believed that protein sliding, hopping, and transfer between adjacent DNA segments, during which the protein nonspecifically interacts with DNA, are central to the speed of their specific recognition. In this study, we focused on the structural and dynamic features of proteins when they scan the DNA. Using a simple computational model that represents protein-DNA interactions by electrostatic forces, we identified that the protein makes use of identical binding interfaces for both nonspecific and specific DNA interactions. Accordingly, in its one-dimensional diffusion along the DNA, the protein is bound at the major groove and performs a helical motion, which is stochastic and driven by thermal diffusion. A microscopic structural insight into sliding from our model, which is governed by electrostatic forces, corroborates previous experimental studies suggesting that the active site of some regulatory proteins continually faces the interior of the DNA groove while sliding along sugar-phosphate rails. The diffusion coefficient of spiral motion along the major groove of the DNA is not affected by salt concentration, but the efficiency of the search can be significantly enhanced by increasing salt concentration due to a larger number of hopping events. We found that the most efficient search comprises ∼ 20% sliding along the DNA and ∼ 80% hopping and three-dimensional diffusion. The presented model that captures various experimental features of facilitated diffusion has the potency to address other questions regarding the nature of DNA search, such as the sliding characteristics of oligomeric and multidomain DNA-binding proteins that are ubiquitous in the cell.     

Phosphorylation-induced rearrangement of the histone H3 NH2-terminal domain during mitotic chromosome condensation

The NH2-terminal domain (N-tail) of histone H3 has been implicated in chromatin compaction and its phosphorylation at Ser10 is tightly correlated with mitotic chromosome condensation. We have developed one mAb that specifically recognizes histone H3 N-tails phosphorylated at Ser10 (H3P Ab) and another that recognizes phosphorylated and unphosphorylated H3 N-tails equally well (H3 Ab). Immunocytochemistry with the H3P Ab shows that Ser10 phosphorylation begins in early prophase, peaks before metaphase, and decreases during anaphase and telophase. Unexpectedly, the H3 Ab shows stronger immunofluorescence in mitosis than interphase, indicating that the H3 N-tail is more accessible in condensed mitotic chromatin than in decondensed interphase chromatin. In vivo ultraviolet laser cross-linking indicates that the H3 N-tail is bound to DNA in interphase cells and that binding is reduced in mitotic cells. Treatment of mitotic cells with the protein kinase inhibitor staurosporine causes histone H3 dephosphorylation and chromosome decondensation. It also decreases the accessibility of the H3 N-tail to H3 Ab and increases the binding of the N-tail to DNA. These results indicate that a phosphorylation-dependent weakening of the association between the H3 N-tail and DNA plays a role in mitotic chromosome condensation.     

Sliding of Proteins Non-specifically Bound to DNA: Brownian Dynamics Studies with Coarse-Grained Protein and DNA Models

DNA binding proteins efficiently search for their cognitive sites on long genomic DNA by combining 3D diffusion and 1D diffusion (sliding) along the DNA. Recent experimental results and theoretical analyses revealed that the proteins show a rotation-coupled sliding along DNA helical pitch. Here, we performed Brownian dynamics simulations using newly developed coarse-grained protein and DNA models for evaluating how hydrodynamic interactions between the protein and DNA molecules, binding affinity of the protein to DNA, and DNA fluctuations affect the one dimensional diffusion of the protein on the DNA. Our results indicate that intermolecular hydrodynamic interactions reduce 1D diffusivity by 30%. On the other hand, structural fluctuations of DNA give rise to steric collisions between the CG-proteins and DNA, resulting in faster 1D sliding of the protein. Proteins with low binding affinities consistent with experimental estimates of non-specific DNA binding show hopping along the CG-DNA. This hopping significantly increases sliding speed. These simulation studies provide additional insights into the mechanism of how DNA binding proteins find their target sites on the genome.     

The N-terminal Tails of Histones H2A and H2B Adopt Two Distinct Conformations in the Nucleosome with Contact and Reduced Contact to DNA

The nucleosome comprises two histone dimers of H2A-H2B and one histone tetramer of (H3-H4)₂, wrapped around by ~145 bp of DNA. Detailed core structures of nucleosomes have been established by X-ray and cryo-EM, however, histone tails have not been visualized. Here, we have examined the dynamic structures of the H2A and H2B tails in 145-bp and 193-bp nucleosomes using NMR, and have compared them with those of the H2A and H2B tail peptides unbound and bound to DNA. Whereas the H2A C-tail adopts a single but different conformation in both nucleosomes, the N-tails of H2A and H2B adopt two distinct conformations in each nucleosome. To clarify these conformations, we conducted molecular dynamics (MD) simulations, which suggest that the H2A N-tail can locate stably in either the major or minor grooves of nucleosomal DNA. While the H2B N-tail, which sticks out between two DNA gyres in the nucleosome, was considered to adopt two different orientations, one toward the entry/exit side and one on the opposite side. Then, the H2A N-tail minor groove conformation was obtained in the H2B opposite side and the H2B N-tail interacts with DNA similarly in both sides, though more varied conformations are obtained in the entry/exit side. Collectively, the NMR findings and MD simulations suggest that the minor groove conformer of the H2A N-tail is likely to contact DNA more strongly than the major groove conformer, and the H2A N-tail reduces contact with DNA in the major groove when the H2B N-tail is located in the entry/exit side.     

Search by proteins for their DNA target site: 1. The effect of DNA conformation on protein sliding

The recognition of DNA-binding proteins (DBPs) to their specific site often precedes by a search technique in which proteins slide, hop along the DNA contour or perform inter-segment transfer and 3D diffusion to dissociate and re-associate to distant DNA sites. In this study, we demonstrated that the strength and nature of the non-specific electrostatic interactions, which govern the search dynamics of DBPs, are strongly correlated with the conformation of the DNA. We tuned two structural parameters, namely curvature and the extent of helical twisting in circular DNA. These two factors are mutually independent of each other and can modulate the electrostatic potential through changing the geometry of the circular DNA conformation. The search dynamics for DBPs on circular DNA is therefore markedly different compared with linear B-DNA. Our results suggest that, for a given DBP, the rotation-coupled sliding dynamics is precluded in highly curved DNA (as well as for over-twisted DNA) because of the large electrostatic energy barrier between the inside and outside of the DNA molecule. Under such circumstances, proteins prefer to hop in order to explore interior DNA sites. The change in the balance between sliding and hopping propensities as a function of DNA curvature or twisting may result in different search efficiency and speed.     

The amino-terminal tails of histones H2A and H3 coordinate efficient base excision repair,DNA damage signaling and postreplication repair in Saccharomyces cerevisiae

Histone amino-terminal tails (N-tails) are required for cellular resistance to DNA damaging agents; therefore, we examined the role of histone N-tails in regulating DNA damage response pathways in Saccharomyces cerevisiae. Combinatorial deletions reveal that the H2A and H3 N-tails are important for the removal of MMS-induced DNA lesions due to their role in regulating the basal and MMS-induced expression of DNA glycosylase Mag1. Furthermore, overexpression of Mag1 in a mutant lacking the H2A and H3 N-tails rescues base excision repair (BER) activity but not MMS sensitivity. We further show that the H3 N-tail functions in the Rad9/Rad53 DNA damage signaling pathway, but this function does not appear to be the primary cause of MMS sensitivity of the double tailless mutants. Instead, epistasis analyses demonstrate that the tailless H2A/H3 phenotypes are in the RAD18 epistasis group, which regulates postreplication repair. We observed increased levels of ubiquitylated PCNA and significantly lower mutation frequency in the tailless H2A/H3 mutant, indicating a defect in postreplication repair. In summary, our data identify novel roles of the histone H2A and H3 N-tails in (i) regulating the expression of a critical BER enzyme (Mag1), (ii) supporting efficient DNA damage signaling and (iii) facilitating postreplication repair.     

Energetic and structural considerations for the mechanism of protein sliding along DNA in the nonspecific BamHI-DNA complex

The molecular mechanism by which DNA-binding proteins find their specific binding sites is still unclear. To gain insights into structural and energetic elements of this mechanism, we used the crystal structure of the nonspecific BamHI-DNA complex as a template to study the dominant electrostatic interaction in the nonspecific association of protein with DNA, and the possible sliding pathways that could be sustained by such an interaction. Based on calculations using the nonlinear Poisson-Boltzmann method and Brownian dynamics, a model is proposed for the initial nonspecific binding of BamHI to B-form DNA that differs from that seen in the crystal structure of the nonspecific complex. The model is electrostatically favorable, and the salt dependence as well as other thermodynamic parameters calculated for this model are in good agreement with experimental results. Several residues in BamHI are identified for their important contribution to the energy in the nonspecific binding model, and specific mutagenesis experiments are proposed to test the model on this basis. We show that a favorable sliding pathway of the protein along DNA is helical.     

Insights into nonspecific binding of homeodomains from a structure of MATalpha2 bound to DNA

The 2.1-A resolution crystal structure of the MATalpha2 homeodomain bound to DNA reveals the unexpected presence of two nonspecifically bound alpha2 homeodomains, in addition to the two alpha2 homeodomains bound to canonical alpha2 binding sites. One of the extra homeodomains makes few base-specific contacts, while the other extra homeodomain binds to DNA in a previously unobserved manner. This unusually bound homeodomain is rotated on the DNA, making possible major groove contacts by side-chains that normally do not contact the DNA. This alternate docking may represent one way in which homeodomains sample nonspecific DNA sequences.     

SSB functions as a sliding platform that migrates on DNA via reptation

SSB proteins bind to and control the accessibility of single-stranded DNA (ssDNA), likely facilitated by their ability to diffuse on ssDNA. Using a hybrid single-molecule method combining fluorescence and force, we probed how proteins with large binding site sizes can migrate rapidly on DNA and how protein-protein interactions and tension may modulate the motion. We observed force-induced progressive unraveling of ssDNA from the SSB surface between 1 and 6 pN, followed by SSB dissociation at ～10 pN, and obtained experimental evidence of a reptation mechanism for protein movement along DNA wherein a protein slides via DNA bulge formation and propagation. SSB diffusion persists even when bound with RecO and at forces under which the fully wrapped state is perturbed, suggesting that even in crowded cellular conditions SSB can act as a sliding platform to recruit and carry its interacting proteins for use in DNA replication, recombination and repair.     

Isolating Contributions from Intersegmental Transfer to DNA Searching by Alkyladenine DNA Glycosylase

Large genomes pose a challenge to DNA repair pathways because rare sites of damage must be efficiently located from among a vast excess of undamaged sites. Human alkyladenine DNA glycosylase (AAG) employs nonspecific DNA binding interactions and facilitated diffusion to conduct a highly redundant search of adjacent sites. This ensures that every site is searched, but could be a detriment if the protein is trapped in a local segment of DNA. Intersegmental transfer between DNA segments that are transiently in close proximity provides an elegant solution that balances global and local searching processes. It has been difficult to detect intersegmental transfer experimentally; therefore, we developed biochemical assays that allowed us to observe and measure the rates of intersegmental transfer by AAG. AAG has a flexible amino terminus that tunes its affinity for nonspecific DNA, but we find that it is not required for intersegmental transfer. As AAG has only a single DNA binding site, this argues against the bridging model for intersegmental transfer. The rates of intersegmental transfer are strongly dependent on the salt concentration, supporting a jumping mechanism that involves microscopic dissociation and capture by a proximal DNA site. As many DNA-binding proteins have only a single binding site, jumping may be a common mechanism for intersegmental transfer.     

Target search of N sliding proteins on a DNA

At low to moderate ambient salt concentrations, DNA-binding proteins bind relatively tightly to DNA, and only very rarely detach. Intersegmental transfer due to DNA-looping can be excluded by applying an external pulling force to the DNA molecule. Under such conditions, we explore the targeting dynamics of N proteins sliding diffusively along DNA in search of their specific target sequence. At lower densities of binding proteins, we find a reduction of the characteristic search time proportional to N(-2), with corrections at higher concentrations. Rates for detachment and attachment of binding proteins are incorporated in the model. Our findings are in agreement with recent single molecule studies in the presence of bacteriophage T4 gene 32 protein for which the unbinding rate is much lower than the specific binding rate.     

New Insights into Amino-Terminal Translocation as Revealed by the Use of YidC and Sec Depletion Strains

Different attributes of membrane protein substrates have been proposed and characterized as translocation-pathway determinants. However, several gaps in our understanding of the mechanism of targeting, insertion, and assembly of inner-membrane proteins exist. Specifically, the role played by hydrophilic N-terminal tails in pathway selection is unclear. In this study, we have evaluated length and charge density as translocase determinants using model proteins. Strikingly, the 36-residue N-tail of 2Pf3–Lep translocates independent of YidC–Sec. This is the longest known substrate of this pathway. We confirmed this using a newly constructed YidC–Sec double-depletion strain. Increasing its N-tail length with uncharged spacer peptides led to YidC dependence and eventually YidC–Sec dependence, hence establishing that length has a linear effect on translocase dependence. Tails longer than 60 residues were not inserted; however, an MBP–2Pf3–Lep fusion protein could be ranslocated. This suggests that longer N-tails can be translocated if it can engage SecA. In addition, we have examined how the positioning of charges within the translocated N-tail affects the insertion pathway. Additional charges can be translocated by the Lep TM when the charges are distributed across a longer N-tail. We tested charge density as a translocase determinant and confirmed that the addition of positive or negatives charges led to a greater dependence on YidC–Sec when they were placed close to each other than away. Findings from this work make an important advance in our existing knowledge about the different insertion mechanisms of membrane proteins in Escherichia coli.