Dynamic interactions of p53 with DNA in solution by time-lapse atomic force microscopy.

Dynamic interactions of the tumor suppressor protein p53 with a DNA fragment containing a p53-specific recognition sequence were directly observed by time-lapse tapping mode atomic force microscopy (AFM) in liquid. The divalent cation Mg(2+) was used to loosely attach both DNA and p53 to a mica surface so they could be imaged by the AFM while interacting with each other. Various interactions of p53 with DNA were observed, including dissociation/re-association, sliding and possibly direct binding to the specific sequence. Two modes of target recognition of p53 were detected: (a) direct binding, and (b) initial non-specific binding with subsequent translocation by one-dimensional diffusion of the protein along the DNA to the specific site.     

Distinct requirements of adenovirus E1b55K protein for degradation of cellular substrates

The E1b55K and E4orf6 proteins of adenovirus type 5 (Ad5) assemble into a complex together with cellular proteins including cullin 5, elongins B and C, and Rbx1. This complex possesses E3 ubiquitin ligase activity and targets cellular proteins for proteasome-mediated degradation. The ligase activity has been suggested to be responsible for all functions of E1b55K/E4orf6, including promoting efficient viral DNA replication, preventing a cellular DNA damage response, and stimulating late viral mRNA nuclear export and late protein synthesis. The known cellular substrates for degradation by E1b55K/E4orf6 are the Mre11/Rad50/Nbs1 DNA repair complex, the tumor suppressor p53, and DNA ligase IV. Here we show that the degradation of individual targets can occur independently of other substrates. Furthermore, we identify separation-of-function mutant forms of E1b55K that can distinguish substrates for binding and degradation. Our results identify distinct regions of E1b55K that are involved in substrate recognition but also imply that there are additional requirements beyond protein association. These mutant proteins will facilitate the determination of the relevance of specific substrates to the functions of E1b55K in promoting infection and inactivating host defenses.     

Cyclin-Dependent Kinase Inhibitor p21 Modulates the DNA Primer-Template Recognition Complex

The p21 protein, a cyclin-dependent kinase (CDK) inhibitor, is capable of binding to both cyclin-CDK and the proliferating cell nuclear antigen (PCNA). Through its binding to PCNA, p21 can regulate the function of PCNA differentially in replication and repair. To gain an understanding of the precise mechanism by which p21 affects PCNA function, we have designed a new assay for replication factor C (RFC)-catalyzed loading of PCNA onto DNA, a method that utilizes a primer-template DNA attached to agarose beads via biotin-streptavidin. Using this assay, we showed that RFC remains transiently associated with PCNA on the DNA after the loading reaction. Addition of p21 did not inhibit RFC-dependent PCNA loading; rather, p21 formed a stable complex with PCNA on the DNA. In contrast, the formation of a p21-PCNA complex on the DNA resulted in the displacement of RFC from the DNA. The nonhydrolyzable analogs of ATP, adenosine-5′-O-(3-thiotriphosphate) (ATPγS) and adenyl-imidodiphosphate, each stabilized the primer recognition complex containing RFC and PCNA in the absence of p21. RFC in the ATPγS-activated complex was no longer displaced from the DNA by p21. We propose that p21 stimulates the dissociation of the RFC from the PCNA-DNA complex in a process that requires ATP hydrolysis and then inhibits subsequent PCNA-dependent events in DNA replication. The data suggest that the conformation of RFC in the primer recognition complex might change on hydrolysis of ATP. We also suggest that the p21-PCNA complex that remains attached to DNA might function to tether cyclin-CDK complexes to specific regions of the genome.     

Role of cysteine62 in DNA recognition by the P50 subunit of NF-kappa B.

A powerful chemical modification procedure has been developed to define determinants of DNA recognition by the p50 subunit of NF-kappa B. Differential labelling with [14C] iodoacetate has identified a conserved cysteine residue, Cys62, that was protected from modification by the presence of an oligonucleotide containing the specific recognition site of the protein. To determine the importance of this cysteine residue, each of the conserved cysteines in p50 was changed to serine and the DNA binding properties of the mutant proteins determined. Scatchard analysis indicated that the C62S mutant bound to its DNA recognition site with a 10-fold larger dissociation constant than the wild type protein, while the other two mutants bound with an intermediate affinity. Dissociation rate constant measurements correlated well with the dissociation constants for the wild type, C119S, and C273S p50 proteins, whereas the p50 C62S-DNA complex dissociated anomalously quickly. Competition analyses with oligonucleotide variants of the DNA recognition site and nonspecific E. coli DNA revealed that the C62S p50 mutant had an altered DNA binding site specificity and was impaired in its ability to discriminate between specific and non-specific DNA. Thus the sulphydryl group of Cys62 is an important determinant of DNA recognition by the p50 subunit of NF-kappa B.     

Synthesis and testing of a triaza-cyclopenta[b]phenanthrene scaffold as a DNA binding agent

A novel DNA binding agent based upon a triaza-cyclopenta[b]phenanthrene scaffold, compound 1, has been synthesized. dsDNA binding analysis of this compound using the ethidium bromide displacement assay indicated a preference for GC-rich sequences. However, equilibrium dialysis experiments against a variety of nucleic acids showed that the target compound bound about 20-fold tighter to G-quartet DNA than to dsDNA under physiological salt concentrations. The binding of 1 to G-quartet DNA was verified by the ability of the compound to promote the formation of the quartet and to compete with TmPyP4 for binding to the quadruplex. Given the importance of G-quartet binding agents in the treatment of cancer and in the understanding of drug-DNA interactions, 1 and its related analogs should find utility as a new class of G-quartet specific agents.     

Structural mechanism of DNA recognition by the p204 HIN domain

The interferon gamma-inducible protein 16 (IFI16) and its murine homologous protein p204 function in non-sequence specific dsDNA sensing; however, the exact dsDNA recognition mechanisms of IFI16/p204, which harbour two HIN domains, remain unclear. In the present study, we determined crystal structures of p204 HINa and HINb domains, which are highly similar to those of other PYHIN family proteins. Moreover, we obtained the crystal structure of p204 HINab domain in complex with dsDNA and provided insights into the dsDNA binding mode. p204 HINab binds dsDNA mainly through α2 helix of HINa and HINb, and the linker between them, revealing a similar HIN:DNA binding mode. Both HINa and HINb are vital for HINab recognition of dsDNA, as confirmed by fluorescence polarization assays. Furthermore, a HINa dimerization interface was observed in structures of p204 HINa and HINab:dsDNA complex, which is involved in binding dsDNA. The linker between HINa and HINb reveals dynamic flexibility in solution and changes its direction at ∼90° angle in comparison with crystal structure of HINab:dsDNA complex. These structural information provide insights into the mechanism of DNA recognition by different HIN domains, and shed light on the unique roles of two HIN domains in activating the IFI16/p204 signaling pathway.     

The α-Helical Region in p24γ2 Subunit of p24 Protein Cargo Receptor Is Pivotal for the Recognition and Transport of Glycosylphosphatidylinositol-anchored Proteins

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are group of proteins that depend on p24 cargo receptors for their transport from the endoplasmic reticulum to the Golgi apparatus. The GPI anchor is expected to act as a sorting and transport signal, but so far little is known about the recognition mechanism. In the present study we investigate the GPI-AP transport in cell knockdown of p24γ, the most diverse p24 subfamily. Knockdown of p24γ₂ but not of other p24γ family members impaired the transport of a reporter GPI-AP. Restoration of the knockdown-induced phenotype using chimeric constructs between p24γ₂ and the related p24γ₁ further implied a role of the α-helical region of p24γ₂ but not its GOLD domain in the specific binding of GPI-APs. We conclude that motifs in the membrane-adjacent α-helical region of p24γ₂ are involved in recognition of GPI-APs and are consequently responsible for the incorporation of these proteins into coat protein complex II-coated transport vesicles.     

The Rep protein binding elements of the plasmid ColE2-P9 replication origin

The plasmid ColE2-P9 (ColE2) origin (32bp) is specifically recognized by the plasmid-specified Rep protein that initiates DNA replication. The ColE2 origin is divided into at least three functional subregions (I, II, and III), and three sites (a, b, and c) found in subregions I and II play important roles in Rep protein binding. We performed SELEX experiments of plasmid ColE2 to determine the optimal sequences for specific binding of the Rep protein. From these experiments, we obtained a common 16-bp sequence (5'-TGAGACCANATAAGCC-3'), which corresponds to about one half of the minimal ColE2 origin and contains sites a and b. Gel mobility shift assays using single-point mutant origins and the Rep protein further indicated that high affinity sequence-specific recognition by the Rep protein requires sites a, b, and c, but that mutations in site c were less disruptive to this recognition than those in sites a and b.     

Relationship between folding and function in a sequence-specific miniature DNA-binding protein

Previously, we have described a miniature protein-based approach to the design of molecules that bind DNA or protein surfaces with high affinity and specificity. In this approach, the small, well-folded protein avian pancreatic polypeptide acts as a scaffold to present and stabilize an alpha-helical or PPII-helical recognition epitope. The first miniature protein designed in this way, a molecule called p007, presents the alpha-helical recognition epitope found on the bZIP protein GCN4 and binds DNA with nanomolar affinity and exceptional specificity. In this work we use alanine-scanning mutagenesis to explore the contributions of 29 p007 residues to DNA affinity, specificity, and secondary structure. Virtually every residue within the p007 alpha-helix, and most residues within the p007 PPII helix, contribute to both DNA affinity and specificity. These residues include those introduced to make specific and nonspecific DNA contacts, as well as those that complete the miniature protein core. Moreover, there exists a direct correlation between the affinity of a p007 variant for specific DNA and the ability of that variant to select for specific DNA over nonspecific DNA. Although we observe no correlation between alpha-helicity and affinity, we observe a limited correlation between alpha-helicity and sequence specificity that emphasizes the role of coupled binding/folding in the function of p007. Our results imply that formation of a highly evolved set of protein.DNA contacts in the context of a well-packed hydrophobic core, and not the extent of intrinsic alpha-helical structure, is the primary determinant of p007 function.     

Detection of bulky DNA lesions: DDB2 at the interface of chromatin and DNA repair in eukaryotes

Bulky DNA damage is corrected by the nucleotide excision repair (NER) pathway. Although the core biochemical mechanism of NER is understood, details including lesion recognition and repair in the context of chromatin remain to be elucidated. As more data become available, the complexity of lesion recognition in chromatin is becoming clear. This review will discuss current knowledge of DNA damage recognition in the context of chromatin, with a focus on the roles of chromatin remodeling and the specific lesion recognition protein DDB2 (DNA damage-binding protein 2) in chromatin repair. Additionally, we propose a model that ubiquitination-mediated DDB2 dissociation from chromatin, not its degradation, is important for GG-NER progression.     

Synthesis and biological evaluation of 4β-sulphonamido and 4β-[(4'-sulphonamido)benzamide]podophyllotoxins as DNA topoisomerase-IIα and apoptosis inducing agents

A series of new 4β-sulphonamido and 4β-[(4'-sulphonamido)benzamide] conjugates of podophyllotoxin (11a-j and 15a-g) were synthesized and evaluated for anticancer activity against six human cancer cell lines and found to be more potent than etoposide. Some of the compounds 11b, 11d and 11e that showed significant antiproliferative activity in Colo-205 cells, were superior to etoposide. The flow cytometric analysis indicates that these compounds (11b, 11d and 11e) showed G2/M cell cycle arrest and among them 11e is the most effective. It is observed that this compound (11e) caused both single-strand DNA breaks as observed by comet assay as well as double-strand DNA breaks as indicated by γ-H2AX. Further 11e showed inhibition of topo-IIα as observed from Western blot analysis and related studies. Compounds caused activation of ATM as well as Chk1 protein indicating that the compound caused effective DNA damage. Moreover activation of caspase-3, p21, p16, NF-kB and down regulation of Bcl-2 protein suggests that this compound (11e) has apoptotic cell death inducing ability, apart from acting as a topo-IIα inhibitor.     

Alpha-helical linker of an artificial 6-zinc finger peptide contributes to selective DNA binding to a discontinuous recognition sequence

Although many zinc finger motifs have been developed to recognize specific DNA triplets, a rational way to selectively skip a particular non-recognized gap in the DNA sequence has never been established. We have now created a 6-zinc finger peptide with an alpha-helix linker, Sp1ZF6(EAAAR)4, which selectively binds to the discontinuous recognition sites in the same phase (10 bp gap) against the opposite phase (5 bp gap) of the DNA helix. The linker peptide (EAAAR)4 forms an alpha-helix structure stabilized by salt bridges, and the helical length is estimated to be about 30 A, corresponding to that of the 10 bp DNA. The gel shift assays demonstrate that Sp1ZF6(EAAAR)4 preferably binds to the 10 bp-gapped target rather than the 5 bp-gapped target. The CD spectra show that the alpha-helical content of the (EAAAR)4 linker is higher in the complex with the 10 bp-gapped target than in the complex with the 5 bp-gapped target. The present results indicate that the alpha-helical linker is suitable for binding to the recognition sites in the same phase and that the linker induces the loss of binding affinity to the recognition sites with the opposite phase. The engineering of a helix-structured linker in the 6-zinc finger peptides should be one of the most promising approaches for selectively targeting discontinuous recognition sites depending on their phase situations.     

Novel DNA photocleaving agents with high DNA sequence specificity related to the antibiotic bleomycin A2.

We have designed and synthesized a series of novel DNA photocleaving agents which break DNA with high sequence specificity. These compounds contain the non-diffusible photoactive p-nitrobenzoyl group covalently linked via a dimethylene (or tetramethylene) spacer to thiazole analogues of the DNA binding portion of the antibiotic bleomycin A2. By using a variety of 5' or 3' 32P-end labeled restriction fragments from plasmid pBR322 as substrate, we have shown that photoactive bithiazole compounds bind DNA at the consensus sequence 5'-AAAT-3' and induce DNA cleavage 3' of the site. Analysis of cleavage sites on the complementary DNA strand and inhibition of DNA breakage by distamycin A indicates these bithiazole derivatives bind and attack the minor groove of DNA. A photoactive unithiazole compound was less specific inducing DNA breakage at the degenerate site 5'-(A/T)(AA/TT)TPu(A/T)-3'. DNA sequence recognition of these derivatives appears to be determined by the thiazole moiety rather than the p-nitrobenzoyl group: use of a tetramethylene group in place of a dimethylene spacer shifted the position of DNA breakage by one base pair. Moreover, much less specific DNA photocleavage was observed for a compound in which p-nitrobenzoyl was linked to the intercalator acridine via a sequence-neutral hexamethylene spacer. The 5'-AAAT-3' specificity of photoactive bithiazole derivatives contrasts with that of bleomycin A2 which cleaves DNA most frequently at 5'-GPy-3' sequences. These results suggest that the cleavage specificity exhibited by bleomycin is not simply determined by its bithiazole/sulphonium terminus, and the contributions from other features, e.g. its metal-chelating domain, must be considered. The novel thiazole-based DNA cleavage agents described here should prove useful as reagents for probing DNA structure and for elucidating the molecular basis of DNA recognition by bleomycin and other ligands.     

Signals at the bacteriophage phi 29 DNA replication origins required for protein p6 binding and activity.

Protein p6 of Bacillus subtilis phage phi 29 binds specifically to the ends of the viral DNA that contain the replication origins, giving rise to a nucleoprotein structure. DNA regions recognized by protein p6 have been mapped by deletion analysis and DNase I footprinting. Main protein p6-recognition signals have been located between nucleotides 62 and 125 at the right phi 29 DNA end and between nucleotides 46 and 68 at the left end. In addition, recognition signals are also present at other sites within 200-300 bp at each phi 29 DNA end. Protein p6 does not seem to recognize a specific sequence in the DNA, but rather a structural feature, which could be bendability. The formation of the protein p6-DNA nucleoprotein complex is likely to be the structural basis for the protein p6 activity in the initiation of replication.     

Relation between changes in the palindromic fraction and DNA replication during early stages of sea urchin development

Inverted repeat DNA sequences during embryogenesis were tested by comparing the bulk inverted repeat taken from Strongylocentrotus intermedius sea urchin embryos at different stages of development. This fraction exhibited quantitative and qualitative changes. A reversible quantitative decrease was associated with the 16-cell embryo and blastula stages. Sizing on 1.5% agarose gel indicated that the length of the palindromic sequences at the early blastula stage was predominantly about 200 b. p., and at the pluteus stage 240 b. p. Sensitivity of the palindromic sequences to S1 nuclease digestion at the blastula and gastrula stages was different. It was shown that a specific set of the inverted repeats was included in fragments of DNA--comparising the origin of replication. The results suggest that the change of inverted repeats may be determined by replication processes.