Levels of residual structure in disordered interaction domains determine in vitro bindingaffinities, but whether they exert similar roles in cells is not known. Here, we show that increasing residual p53 helicity results in stronger Mdm2 binding, altered p53 dynamics, impaired target gene expression and failure to induce cell cycle arrest upon DNA damage.These results establish that residual structure is an important determinant of signaling fidelity in cells.Intrinsically disordered protein domains often mediate protein-protein interactions that result in disorder-to-order transitions via coupled folding and binding reactions 1 . In addition, many disordered interaction domains exhibit defined levels of transient secondary structure resembling their complex-bound states when free in solution 2 . These levels of residual structure affect binding energies, also by reducing the loss of conformational entropy associated with disorderto-order transitions 3 . Accordingly, higher levels of residual structure in disordered interaction domains increase in vitro binding affinities 4,5 . Here we ask to what extent residual structure contributes to protein binding affinities in cells and whether engineered changes to residual structure affect protein function at the cellular level. To answer these questions, we designed p53 mutants with higher residual helicity within their disordered, N-terminal transactivation domains (TADs) and investigated their effects on cellular Mdm2 binding and p53's ability to induce 2 target gene expression and cell cycle arrest. p53 is activated by many forms of cellular stress, including DNA double-strand breaks (DSBs) and functions as a major tumor suppressor and cell cycle regulator 6 . In the absence of DNA damage, cellular p53 levels are kept low by targeted proteasomal degradation mediated by the E3 ubiquitin ligase Mdm2, which interacts with p53TAD and subsequently ubiquitinates p53's C-terminal regulatory domain 7 . Upon DNA damage, post-translational modifications of p53TAD and Mdm2, together with Mdm2 degradation, disrupt the p53-Mdm2 complex. This leads to p53 accumulation and the expression of p53 target genes that regulate DNA repair, cell cycle arrest, senescence or apoptosis 8,9 . One of these target genes is Mdm2, whose expression establishes a negative feedback loop that shapes cellular p53 dynamics and thereby controls cell fate decisions 10 .In its free form, p53TAD exists in equilibrium between disordered and partially helical conformations 11-13 , whereas residues 19-25 form a stable amphipathic α-helix in the Mdm2 complex 14 (Fig. 1a). To increase the binding affinity between p53 and Mdm2 without altering the binding interface, we designed p53TAD mutants with higher levels of residual helicity by mutating conserved proline residues flanking the Mdm2 binding site (i.e., Pro12, Pro13 or Pro27) to alanines (Supplementary Results, Supplementary Fig. 1a). Using NMR spectroscopy, we determined that wild-type (WT) p53TAD helicity (28%) increased to 64% when we replaced Pro27 with ...
Transposons are natural gene delivery vehicles. The Sleeping Beauty (SB) transposon shows efficient transposition and long-term transgene expression in the cells of vertebrates including humans. SB transposition into chromosomal DNA occurs in a fairly random manner. This is clearly not desirable in human gene therapeutic applications because there are potential genotoxic effects associated with transposon integration. In this study we set out to manipulate the selection of SB's target sites for targeted transposition into predetermined chromosomal regions. We evaluated experimental strategies based on engineered proteins composed of DNA-binding domains fused to (i) the transposase; (ii) another protein that binds to a specific DNA sequence within the transposable element; and (iii) another protein that interacts with the transposase. We demonstrated targeted transposition into endogenous matrix attachment regions (MARs) and a chromosomally integrated tetracycline response element (TRE) in cultured human cells, using targeting proteins that bind to the transposon DNA. An approach based on interactions between the transposase and a targeting protein containing the N-terminal protein interaction domain of SB was found to enable an approximately 10(7)-fold enrichment of transgene insertion at a desired locus. Our experiments provide proof-of-principle for targeted chromosomal transposition of an otherwise randomly integrating transposon. Targeted transposition can be a powerful technology for safe transgene integration in human therapeutic applications.
The Sleeping Beauty (SB) element is a useful tool to probe transposon-host interactions in vertebrates. We investigated requirements of DNA repair factors for SB transposition in mammalian cells. Factors of nonhomologous end joining (NHEJ), including Ku, DNA-PKcs, and Xrcc4 as well as Xrcc3/Rad51C, a complex that functions during homologous recombination, are required for efficient transposition. NHEJ plays a dominant role in repair of transposon excision sites in somatic cells. Artemis is dispensable for transposition, consistent with the lack of a hairpin structure at excision sites. Ku physically interacts with the SB transposase. DNA-PKcs is a limiting factor for transposition and, in addition to repair, has a function in transposition that is independent from its kinase activity. ATM is involved in excision site repair and affects transposition rates. The overlapping but distinct roles of repair factors in transposition and in V(D)J recombination might influence the outcomes of these mechanistically similar processes.
Using the yeast artificial chromosome (YAC) 116 flanking the autosomal recessive spinal muscular atrophy (SMA) gene region, we have screened a human fetal brain cDNA library and isolated the cDNA clone 14-3/9 with an insert size of 2.5 kb. The cDNA clone could be identified as part of the human rRNA gene coding for 28S rRNA with a total size of 5025 bp. The human 28S rRNA is involved in the organization of the 60S ribosomal subparticle and is arranged in a 13-kb pre-rRNA transcription unit that occurs in tandem repeat clusters. Multiple copies of the rRNA gene have been mapped by pulsed field blot hybridization in the YAC contig between YAC 66 and YAC 116, which encompasses the SMA candidate gene, and additionally in the distally localized YAC 153.
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