Higher-order Nucleoprotein Structures Research Articles

Single-Molecule DNA Analysis Reveals That Yeast Hop1 Protein Promotes DNA Folding and Synapsis: Implications for Condensation of Meiotic Chromosomes

During meiosis, long-range interaction between homologous chromosomes is thought to be crucial for homology recognition, exchange of DNA strands, and production of normal haploid gametes. However, little is known about the identity of the proteins involved and the actual molecular mechanism(s) by which chromosomes recognize and recombine with their appropriate homologous partners. Single-molecule analyses have the potential to provide insights into our understanding of this fascinating and long-standing question. Using atomic force microscopy and magnetic tweezers techniques, we discovered that Hop1 protein, a key structural component of Saccharomyces cerevisiae synaptonemal complex, exhibits the ability to bridge noncontiguous DNA segments into intramolecular stem-loop structures in which the DNA segments appear to be fully synapsed within the filamentous protein stems. Additional evidence suggests that Hop1 folds DNA into rigid protein-DNA filaments and higher-order nucleoprotein structures. Importantly, Hop1 promotes robust intra- and intermolecular synapsis between double-stranded DNA molecules, suggesting that juxtaposition of DNA sequences may assist in strand exchange between homologues by recombination-associated proteins. Finally, the evidence from ensemble experiments is consistent with the notion that Hop1 causes rigidification of DNA molecules. These results provide the first direct evidence for long-range protein-mediated DNA-DNA synapsis, independent of crossover recombination, which is presumed to occur during meiotic recombination.

Functional Evolution of Bacterial Histone-Like HU Proteins

Bacterial histone-like HU proteins are critical to maintenance of the nucleoid structure. In addition, they participate in all DNA-dependent functions, including replication, repair, recombination and gene regulation. In these capacities, their function is typically architectural, inducing a specific DNA topology that promotes assembly of higher-order nucleo-protein structures. Although HU proteins are highly conserved, individual homologs have been shown to exhibit a wide range of different DNA binding specificities and affinities. The existence of such distinct specificities indicates functional evolution and predicts distinct in vivo roles. Emerging evidence suggests that HU proteins discriminate between DNA target sites based on intrinsic flexure, and that two primary features of protein binding contribute to target site selection: The extent to which protein-mediated DNA kinks are stabilized and a network of surface salt-bridges that modulate interaction between DNA flanking the kinks and the body of the protein. These features confer target site selection for a specific HU homolog, they suggest the ability of HU to induce different DNA structural deformations depending on substrate, and they explain the distinct binding properties characteristic of HU homologs. Further divergence is evidenced by the existence of HU homologs with an additional lysine-rich domain also found in eukaryotic histone H1.

Structural Aspects of RecA-Dependent Homologous Strand Exchange Involving Human Telomeric DNA

Telomeric DNA sequences in human cells and those of other vertebrates consist of long d(TTAGGG) repeats. In somatic cells, telomeres shorten every cell division with shortening serving as a mitotic clock that counts cell divisions and ultimately results in cellular senescence. Telomere length is principally maintained by a ribonucleoprotein, telomerase. However, a non-negligible proportion of human cells use a recombination-based mechanism for telomere maintenance, termed alternative maintenance of telomeres (ALT). Although the molecular mechanism of ALT is not known, GT-rich sequences in prokaryotes and eukaryotes display high levels of recombination relative to those of non-GT-rich DNA. We show that human telomeric strand-exchange complexes mediated by Escherichia coli RecA protein differ from those formed with nontelomeric sequences. Moreover, telomeric strand-exchange intermediates, unlike those involving nontelomeric sequences, exhibit a tendency to form higher-order nucleoprotein structures. We propose that the strong DNA unwinding activity inherent in the assembly of the RecA strand-exchange complex promotes the formation of alternative DNA structures at human telomeric loci. Organization of these noncanonical structures into higher-order complexes involving multiple DNA duplexes could facilitate the search for homology on different DNA molecules and provide a framework for understanding recombination-dependent mechanisms of telomere maintenance.

A mini review of the high mobility group proteins of insects.

High mobility group (HMG) proteins are an abundant class of chromosomal proteins facilitate assembly of higher order structures. The mammalian HMG proteins have been grouped into three distinct families on the basis of their characteristic functional sequence: the HMGB, the HMGN, and the HMGA family. The HMG proteins of Drosophila melanogaster and Chironomus tentans are the best characterized dipteran insect HMG proteins. Three abundant members of this group of nonhistone proteins were detected in those insects. Two of them belong to the HMGB family and one to the HMGA family. The possible relatedness of these proteins to the formation of higher order nucleoprotein structures and their possible role in the regulation of transcription is discussed.

Biophysical Analysis of Specific Genomic Loci Assembled as Chromatin In Vivo

Publisher Summary This chapter focuses on the recently adapted technique of agarose multigel electrophoresis (AME) for analysis of the higher-order nucleoprotein structure of specific genomic loci that have been isolated as native chromatin in unfractionated low-salt nuclear extracts. It also describes how AME can be used as a biophysical method for characterizing specific in vivo –assembled chromatin fragments. The general concepts presented in this chapter are based on the recent studies of genomic murine mammary tumor virus (MMTV) promoters. This approach yields analytical measurements of average macromolecular radii and surface charge density that in turn allows one to evaluate the condensation behavior and conformational flexibility of the chromatin fragment being studied. AME is an easily accessible method that is performed with the commercially available electrophoresis apparatus. In an AME experiment, the sample of interest is first spiked with the spherical bacteriophage T3. The only aspect of the AME approach that is not general is obtaining the specific genomic chromatin fragment of interest in a low-salt nuclear extract. As with any newly introduced technical approach, the ability to accurately interpret AME data have capability to increase in direct proportion to the number of systems that are characterized by this method in the future.

Use of site-specific recombination as a probe of nucleoprotein complex formation in chromatin.

DNA transactions in eukaryotes require that proteins gain access to target sequences packaged in chromatin. Further, interactions between distinct nucleoprotein complexes are often required to generate higher-order structures. Here, we employed two prokaryotic site-specific recombination systems to investigate how chromatin packaging affects the assembly of nucleoprotein structures of different complexities at more than 30 genomic loci. The dynamic nature of chromatin permitted protein-DNA and DNA-DNA interactions for sites of at least 34 bp in length. However, the assembly of higher-order nucleoprotein structures on targets spanning 114 bp was impaired. This impediment was maintained over at least 72 h and was not affected by the transcriptional status of chromatin nor by inhibitors of histone deacetylases and topoisomerases. Our findings suggest that nucleosomal linker-sized DNA segments become accessible within hours for protein binding due to the dynamic nature of chromatin. Longer segments, however, appear refractory for complete occupancy by sequence-specific DNA-binding proteins. The results thus also provide an explanation why simple recombination systems such as Cre and Flp are proficient in eukaryotic chromatin.

Interaction of the Escherichia coli DnaA protein with bacteriophage lambda DNA.

Interaction of the Escherichia coli DnaA (replication initiator) protein with restriction fragments of phage lambda DNA demonstrated differential binding of DnaA along the whole lambda DNA. Interaction of DnaA with the lambda replication region (from the promoter pR to the origin of replication, orilambda) demonstrated a strong binding of DnaA to the region around the p(o) promoter where synthesis of a short antisense oop RNA is initiated. The four sequences protected by DnaA (two 9mers and two 5mers) are not related even to a relaxed DnaA box. The pattern of protection of these four sequences and the location of three DNase I hypersensitive sites in the lambda DNA r strand, together with results of mobility shift assays and electron microscopy studies, may indicate an interaction involving DnaA monomers bound to different DNA positions on one side of the helix and the formation of higher-order nucleoprotein structures. Therefore, it is tempting to suggest that DnaA, in addition to its activity in regulation of replication and transcription, could be considered as a factor which structures certain chromosomal regions.

Chromatin structure analysis of the human c-fos promoter reveals a centrally positioned nucleosome.

Chromatin architecture plays an important regulatory role in nuclear transcription in eukaryotes. In particular, a preset chromatin organization has been proposed to prime rapidly inducible genes for response to extracellular signals. We analyzed the chromatin architecture of the rapidly inducible human c-fos gene using a combination of nuclease digestion studies. Several regions of nuclease sensitivity in the c-fos gene are observed: (i) a hypersensitive site at position -1900; (ii) a region centered at -350 that corresponds to the locations of the serum response element (SRE) and the sis-inducible element (SIE); (iii) a region around the transcriptional start site that includes the TATA box, the cAMP-responsive element (CRE) and a directly repeated sequence (DRE); and (iv) two sites at positions +250 and +550 that appear to delineate the linker regions of two positioned nucleosomes. In contrast, the region from -280 to -90 is strongly resistant to nuclease digestion. We identify a highly positioned nucleosome in this region of the c-fos promoter that may contribute to a defined higher-order nucleoprotein structure containing the nucleosome and proteins bound to the SIE, SRE, DRE, and CRE elements and TATA box. This structure may lead to a functional juxtaposition of the regulatory elements of the c-fos promoter and suggests that such a chromatin architecture is particularly suited for presetting promoters for rapid responsiveness.

The replisome organizer (G38P) of Bacillus subtilis bacteriophage SPP1 forms specialized nucleoprotein complexes with two discrete distant regions of the SPP1 genome

Initiation of Bacillus subtilis bacteriophage SPP1 DNA replication requires the products of genes 38, 39 and 40 (G38P, G39P and G40P). G38P specifically binds two discrete regions, which are 32.1 kb apart in a linear map of the SPP1 genome. One of these target sites, which maps at the left end of the phage genome, within gene 38, was shown to function as an origin of replication and was therefore termed left origin ( oriL). The other site, which lies within a non-coding segment in the late transcribed region on the right end of the genome, was termed oriR. Both sites contain two types of repeated elements (termed Box AB and A + T-rich region). The K app for the G38P- oriL DNA and G38P- oriR DNA complexes was estimated to be 1 nM and 4 nM, respectively. G38P binds to the distant oriL and oriR sites cooperatively. DNase I footprinting experiments showed protection by G38P in Box AB, but not in the A + T-rich region. Electron microscopy analysis showed that G38P forms a higher-order nucleoprotein structure with the SPP1 oriL and oriR sites through protein-protein interaction. G38P binding at its cognate sites does not seem to modify the length of the DNA, but to bend it. These results suggest that G38P forms a nucleoprotein complex on the regions where the SPP1 replication origins were previously predicted.

Three-Site Synapsis during Mu DNA Transposition: A Critical Intermediate Preceding Engagement of the Active Site

The chemical steps of bacteriophage Mu DNA transposition take place within a higher order nucleoprotein structure. We describe a novel intermediate that precedes the previously characterized transpososomes and directly demonstrates the interaction of a distant enhancer element with recombination regions. The transpositional enhancer interacts with the Mu left and right ends to form a three-site synaptic (LER) complex. Under normal reaction conditions, the LER complex is rapidly converted into the more stable Mu transpososomes. However, mutation of the Mu terminal nucleotides results in accumulation of the LER and a failure to form the type 0 transpososome. During the transition from LER to type 0, the Mu DNA termini and the active site of the transposase engage in a catalytically competent conformation.

The Small Subunit of the Terminase Enzyme of Bacillus subtilisBacteriophage SPP1 forms a Specialized Nucleoprotein Complex with the Packaging Initiation Region

Initiation of SPP1 DNA packaging requires the gene 1and gene 2products (G 1P and G 2P), which are different subunits of the terminase enzyme. G 1P specifically recognizes the phage packaging initiation region ( pac). The apparent equilibrium constant for the G 1P- pac-DNA complex was estimated to be 9 nM. DNase I footprinting experiments reveal that the pacregion can be subdivided into three discrete sites ( pacL, pacC and pacR). G 1P binds co-operatively to the non-adjacent pacL and pacR sites. Several G1P protomers bind to the target sequences which map close to the paccleavage site ( pacC site), but do not overlap with it. G 1P interacts in a different fashion with the encapsidated ( pacR site) and with the non-encapsidated ( pacL site) end of the phage genome. G 1P interaction with the intrinsically bent pacL DNA occurs only on one face of the DNA double helix. G 1P binding to the pacL and in the pacR region results in a DNA loop. Electron microscopy of purified G 1P shows that the protein is an oligomer in solution. G 1P binding to the core region of the pacL site could facilitate the formation of a higher-order nucleoprotein structure. This specialized complex would allow the pacDNA to form a loop between binding sites brought together by interaction with G 1P. The results presented here suggest that G 1P could provide a tool to discriminate the first encapsidated end, which contains pacR, from the non-encapsidated pacL end.

Partition Functions of Mini-F Affect Plasmid DNA Topology in Escherichia coli

Efficient segregation of the low copy number plasmid mini-F is dependent on partition functions encoded by the plasmid sopABCgenes. The sopregion encodes proteins SopA and SopB and a cis-acting element, sopC, which may function as a centromere analog. The SopC segment contains 12 imperfect 43 bp repeats to which the SopB protein binds. We have found that mutations in the sopgenes affect superhelicity of isolated plasmid DNA. Plasmids with mutations in sopBor a deletion of the sopCsegment were more highly negatively supercoiled than normal. In contrast, a mutation in the autoregulatory SopA protein resulted in plasmid DNA that was more relaxed. The SopAB proteins provided in transto a pBR322 plasmid carrying sopCresulted in the relaxation of negative supercoils. We suggest that binding of SopB protein to the cis-acting sopCsegment in vivo, alone or in conjunction with other proteins, produced a change in DNA topology in which positive superhelical turns were3 introduced locally. This higher-order nucleoprotein structure may allo interaction of plasmid mini-F with the partition apparatus.

Higher-order nucleoprotein complexes in transcription: analogies with site-specific recombination

Regulation of transcription involves the assembly of multiprotein complexes at enhancers and promoters. Interactions between adjacent and non-adjacent DNA-binding proteins can augment the specificity and stability of multi-component nucleoprotein complexes. Recently, several proteins have been identified that can function as ‘architectural’ elements in the assembly of higher-order nucleoprotein structures reminiscent of those involved in site-specific recombination in prokaryotes.

Lrp, a major regulatory protein in Escherichia coli, bends DNA and can organize the assembly of a higher-order nucleoprotein structure.

Lrp (Leucine-responsive regulatory protein) is a global regulatory protein that controls the expression of many operons in Escherichia coli. One of those operons, ilvIH, contains six Lrp binding sites located within a several hundred base pair region upstream of the promoter region. Analysis of the binding of Lrp to a set of circularly permuted DNA fragments from this region indicates that Lrp induces DNA bending. The results of DNase I footprinting experiments suggest that Lrp binding to this region facilitates the formation of a higher-order nucleoprotein structure. To define more precisely the degree of bending associated with Lrp binding, one or two binding sites were separately cloned into a pBend vector and analyzed. Lrp induced a bend of approximately 52 degrees upon binding to a single binding site, and the angle of bending is increased to at least 135 degrees when Lrp binds to two adjacent sites. Lrp-induced DNA bending, and a natural sequence-directed bend that exists within ilvIH DNA, may be architectural elements that facilitate the assembly of a nucleoprotein complex.

The HMG domain of lymphoid enhancer factor 1 bends DNA and facilitates assembly of functional nucleoprotein structures

The high mobility group (HMG) domain is a DNA-binding motif that is associated with several eukaryotic regulatory proteins, including the lymphoid enhancer-binding factor LEF-1 and the testis-determining factor SRY. Here, we provide evidence that DNA binding by the HMG domain of LEF-1 involves primarily minor groove contacts and induces a bend of approximately 130° in the DNA helix. Bending was also found to accompany sequence-specific DNA binding by the SRY-HMG domain. Examining possible regulatory roles of HMG domain-induced DNA bends, we found that LEF-1 can function in a manner similar to bacterial integration host factor and facilitate communication between widely separated protein-binding sites in a recombination assay. Together with the previous observation that LEF-1 by itself is unable to augment basal promoter activity, these data suggest that HMG domain proteins can serve as “architectural” elements in the assembly of higher-order nucleoprotein structures.

The interaction of recombination proteins with supercoiled DNA: Defining the role of supercoiling in lambda integrative recombination

Lambda integrative recombination depends on supercoiling of the phage attachment site, attP. Using dimethylsulfate protection and indirect end-labeling, the interaction of the recombination proteins Int and IHF with supercoiled and linear attP has been studied. Supercoiling enhances the binding of Int to attP, but not if a truncated attP site is employed or if IHF is omitted. We reason that the altered affinity reflects the formation of a higher-order nucleoprotein structure, an “ attP intasome,” that involves Int and IHF assembly of both arms of attP into a wrapped configuration. The good correlation between the degree and sign of supercoiling needed to promote recombination and that needed for the “ attP intasome” indicates that the primary role of supercoiling is to drive the formation of the wrapped structure.