Protein motion from non-specific to specific DNA by three-dimensional routes aided by supercoiling

DNA-binding proteins are generally thought to locate their target sites by first associating with the DNA at random and then translocating to the specific site by one-dimensional (1D) diffusion along the DNA. We report here that non-specific DNA conveys proteins to their target sites just as well when held near the target by catenation as when co-linear with the target. Hence, contrary to the prevalent view, proteins move from random to specific sites primarily by three-dimensional (3D) rather than 1D pathways, by multiple dissociation/re-association events within a single DNA molecule. We also uncover a role for DNA supercoiling in target-site location. Proteins find their sites more readily in supercoiled than in relaxed DNA, again indicating 3D rather than 1D routes.     

One- and three-dimensional pathways for proteins to reach specific DNA sites

Proteins that interact with specific DNA sites bind to DNA at random and then translocate to the target site. This may occur by one-dimensional diffusion along the DNA, or through three-dimensional space via multiple dissociation/re-associations. To distinguish these routes, reactions of the ECO:RV endonuclease were studied on substrates with two ECO:RV sites separated by varied distances. The fraction of encounters between the DNA and the protein that resulted in the processive cleavage of both sites decreased as the length of intervening DNA was increased, but not in the manner demanded for one-dimensional diffusion. The variation in processivity with inter-site spacing shows instead that protein moves from one site to another through three-dimensional space, by successive dissociation/re-associations, though each re-association to a new site is followed by a search of the DNA immediately adjacent to that site. Although DNA-binding proteins are usually thought to find their target sites by one-dimensional pathways, three-dimensional routes may be more common than previously anticipated.     

Demethylation initiated by ROS1 glycosylase involves random sliding along DNA

Active DNA demethylation processes play a critical role in shaping methylation patterns, yet our understanding of the mechanisms involved is still fragmented and incomplete. REPRESSOR OF SILENCING 1 (ROS1) is a prototype member of a family of plant 5-methylcytosine DNA glycosylases that initiate active DNA demethylation through a base excision repair pathway. As ROS1 binds DNA non-specifically, we have critically tested the hypothesis that facilitated diffusion along DNA may contribute to target location by the enzyme. We have found that dissociation of ROS1 from DNA is severely restricted when access to both ends is obstructed by tetraloops obstacles. Unblocking any end facilitates protein dissociation, suggesting that random surface sliding is the main route to a specific target site. We also found that removal of the basic N-terminal domain of ROS1 significantly impairs the sliding capacity of the protein. Finally, we show that sliding increases the catalytic efficiency of ROS1 on 5-meC:G pairs, but not on T:G mispairs, thus suggesting that the enzyme achieves recognition and excision of its two substrate bases by different means. A model is proposed to explain how ROS1 finds its potential targets on DNA.     

Facilitated diffusion of a DNA binding protein on chromatin.

Facilitated diffusion accounts for the rapid rate of association of many bacterial DNA binding proteins with specific DNA sequences in vitro. In this mechanism the proteins bind at random to non-specific sites on the DAN and diffuse (by 'sliding' or 'hopping') along the DNA chain until they arrive at their specific functional sites. We have investigated whether such a mechanism can operate in chromatin by using a bacterial DNA binding protein, Escherichia coli RNA polymerase, that depends on linear diffusion to locate initiation sites on DNA. We have measured the competition between chromatin and its free DNA for the formation of initiation complexes. Only the short linker segments exposed by the removal of histone H1 are available for interaction with the polymerase, but the sparsely distributed promoter sites on the linker DNA of such a polynucleosome chain are located at the same rate as those on DNA. We conclude that the polymerase is free to migrate between the separate linker DNA segments of a polynucleosome chain to reach a promoter site. This chain thus permits the 'hopping' of proteins between neighboring linker segments in their search for a target site on the accessible DNA.     

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.     

How to build a centromere: from centromeric and pericentromeric chromatin to kinetochore assembly.

The assembly of the centromere, a specialized region of DNA along with a constitutive protein complex which resides at the primary constriction and is the site of kinetochore formation, has been puzzling biologists for many years. Recent advances in the fields of chromatin, microscopy, and proteomics have shed a new light on this complex and essential process. Here we review recently discovered mechanisms and proteins involved in determining mammalian centromere location and assembly. The centromeric core protein CENP-A, a histone H3 variant, is hypothesized to designate centromere localization by incorporation into centromere-specific nucleosomes and is essential for the formation of a functional kinetochore. It has been found that centromere localization of centromere protein A (CENP-A), and therefore centromere determination, requires proteins involved in histone deacetylation, as well as base excision DNA repair pathways and proteolysis. In addition to the incorporation of CENP-A at the centromere, the formation of heterochromatin through histone methylation and RNA interference is also crucial for centromere formation. The assembly of the centromere and kinetochore is complex and interdependent, involving epigenetics and hierarchical protein-protein interactions.     

A model for the mediation of processivity of DNA-targeting proteins by nonspecific binding: dependence on DNA length and presence of obstacles

A physical and mathematical model is presented to explain processivity of proteins on DNA. In this model, a DNA-targeting protein such as a restriction enzyme can diffuse to the DNA surface and nonspecifically bind to it. Once on the DNA surface it will either move along the DNA or equilibrate with the surrounding region. Owing to the nonspecific binding, the search for a specific site on the DNA occurs in a reduced dimensionality, and the protein appears processive when moving from one specific site to another. The simplest version of this nonspecific-binding-facilitated diffusion model is solved and the results quantitatively explain experimentally observed dependence of the processivity ratio on the intervening DNA length between two specific sites.     

Facilitated diffusion in chromatin lattices: mechanistic diversity and regulatory potential

The interaction between a protein and a specific DNA site is the molecular basis for vital processes in all organisms. Location of the DNA target site by the protein commonly involves facilitated diffusion. Mechanisms of facilitated diffusion vary among proteins; they include one- and two-dimensional sliding along DNA, direct transfer between uncorrelated sites, as well as combinations of these mechanisms. Facilitated diffusion has almost exclusively been studied in vitro. This review discusses facilitated diffusion in the context of the living cell and proposes a theoretical model for facilitated diffusion in chromatin lattices. Chromatin structure differentially affects proteins in different modes of diffusion. The interplay of facilitated diffusion and chromatin structure can determine the rate of protein association with the target site, the frequency of association-dissociation events at the target site, and, under particular conditions, the occupancy of the target site. Facilitated diffusion is required in vivo for efficient DNA repair and bacteriophage restriction and has potential roles in fine-tuning gene regulatory networks and kinetically compartmentalizing the eukaryotic nucleus.     

Ubiquitin-binding domains and their role in the DNA damage response

The modification of eukaryotic proteins by covalent attachment of ubiquitin is a versatile signaling event with a wide range of possible consequences. Canonical poly-ubiquitination by Lys-48 linked chains usually destines a protein for degradation by the proteasome. By contrast, attachment of a single ubiquitin or ubiquitin chains linked through Lys-63 or Lys-6 serves a non-proteolytic role. Over the last years, evidence has accumulated that several nuclear proteins become ubiquitinated in response to DNA damage. Typically, these proteins carry mono-ubiquitin or non-classical ubiquitin chains and are localized close to the site of DNA damage. Of particular interest are PCNA and the variant histone H2AX, two key proteins whose ubiquitination serves to recruit factors needed by the cell to cope with the damage. A prerequisite for docking effector proteins to the site of the lesion is the detection of a specific ubiquitin modification, a process that can be mediated by a range of dedicated ubiquitin-binding domains (UBDs). As the same types of ubiquitin modification are involved in entirely different processes, the recognition of the ubiquitin mark has to go along with the recognition of the modified protein. Thus, ubiquitin-binding domains gain their specificity through combination with other recognition domains and motifs. This review discusses ubiquitin-binding domains relevant to the DNA damage response, including their binding mode, their specificity, and their interdependence with other factors. For several repair pathways, current knowledge of the events downstream of the ubiquitin mark is sketchy. A closer look at orphan UBD proteins might lead to the identification of missing pieces in the DNA response puzzle.     

Comparative analysis of non-random DNA repair following Ac transposon excision in maize and Arabidopsis

Ac/Ds transposable elements often leave short DNA rearrangements, or 'footprints,' at the sites where they excise. Previous studies at the maize waxy ( wx ) gene suggest that the DNA repair that forms transposon footprints is not random. Each excision site consistently displays a different, predominant repair product suggesting flanking DNA may influence footprint formation. We have expanded these studies to show that predominant end-joining products also form in association with Ac/Ds excision in Arabidopsis and that chromosomal location of the Ac -containing construct does not appear to influence this repair. The predominant repair product is identical in both maize and Arabidopsis for Ac elements with the same adjacent DNA sequences. However, a broader range of minor footprint types is observed in Arabidopsis , including footprints that are rare in maize, suggesting potential differences in the host proteins involved in either transposition, repair or both. The data also suggest that the sequences influencing footprint formation are within 39 bp 5' and 18 bp 3' of the transposon. These studies demonstrate that transgenic Ac/Ds -containing plants will be useful tools in dissecting plant DNA repair processes.     

Transport of proteins in eukaryotic cells: more questions ahead

Some newly synthesized proteins contain signals that direct their transport to their final location within or outside of the cell. Targeting signals are recognized by specific protein receptors located either in the cytoplasm or in the membrane of the target organelle. Specific membrane protein complexes are involved in insertion and translocation of polypeptides across the membranes. Often, additional targeting signals are required for a polypeptide to be further transported to its site of function. In this review, we will describe the trafficking of proteins to various cellular organelles (nucleus, chloroplasts, mitochondria, peroxisomes) with emphasis on transport to and through the secretory pathway.     

Strong physical constraints on sequence-specific target location by proteins on DNA molecules

Sequence-specific binding to DNA in the presence of competing non-sequence-specific ligands is a problem faced by proteins in all organisms. It is akin to the problem of parking a truck at a loading bay by the side of a road in the presence of cars parked at random along the road. Cars even partially covering the loading bay prevent correct parking of the truck. Similarly on DNA, non-specific ligands interfere with the binding and function of sequence-specific proteins. We derive a formula for the probability that the loading bay is free from parked cars. The probability depends on the size of the loading bay and allows an estimation of the size of the footprint on the DNA of the sequence-specific protein by assaying protein binding or function in the presence of increasing concentrations of non-specific ligand. Assaying for function gives an 'activity footprint'; the minimum length of DNA required for function rather than the more commonly measured physical footprint. Assaying the complex type I restriction enzyme, EcoKI, gives an activity footprint of approximately 66 bp for ATP hydrolysis and 300 bp for the DNA cleavage function which is intimately linked with translocation of DNA by EcoKI. Furthermore, considering the coverage of chromosomal DNA by proteins in vivo, our theory shows that the search for a specific DNA sequence is very difficult; most sites are obscured by parked cars. This effectively rules out any significant role in target location for mechanisms invoking one-dimensional, linear diffusion along DNA.     

A code governing specific binding of regulatory proteins to DNA and structure of stereospecific sites of regulatory proteins]

A model is proposed for the structure of stereospecific sites in regulatory proteins. On its basis a possible code is suggested that governs the binding of regulatory proteins at specific control sites on DNA. Stereospecific sites of regulatory proteins are assumed to contain pairs of antiparallel polypeptide chain segments which form a right-hand twisted antiparallel beta-sheet, with single-stranded regions at the ends of the beta-structure. The model predicts that binding reaction between a regulatory protein and double-helical DNA is a cooperative phenomenon and is accompanied by significant structural alteration at the stereospecific site of the protein. Half of hydrogen bonds normally existing in beta-structure are broken upon complex formation with DNA and a new set of hydrogen bonds is formed between polypeptide amide groups and DNA base pairs. In a stereospecific site, one chain (t-chain) is attached through hydrogen bonds to the carbonyl oxygens of pyramides and N3 adenines lying in one DNA strand, while the second polypeptide chain (g chain) is hydrogen bonded to the 2-amino groups of guanine residues lying in the opposite DNA strand. The amide groups serve as specific reaction sites being hydrogen bond acceptors in g-chain and hydrogen bond donors in t-chain. The single-stranded portions of t- and g-chains lying in neighbouring subunits of regulatory protein interact with each other forming deformed beta-sheets. The recognition of regulatory sequences by proteins is based on the structural complementarity between stereospecific sites of regulatory proteins and base pairs sequences at the control sites. An essential feature of these sequences is the asymmetrical distribution of guanine residues between the two DNA strands. The code predicts that there are six fundamental amino acid residues (serine, threonine, asparagine, histidine, glutamine and cysteine) whose sequence in stereospecific site determines the base pair sequence to which a given regulatory protein would bind preferentially. The code states a correspondence between four amino acid residues at the stereospecific site of regulatory protein with the two residues being in t- and g-segments, respectively, and AT(GC) base pair at the control site. It is thus possible to determine which amino acid residues in the repressor and which base pairs in the operator DNA are involved in specific interactions with each other, as exemplified by lac repressor binding to lac operator.