The temporal order of replication of DNA sequences in the chromosomal domain containing the human j3-globin gene cluster and its flanking sequences (140 kilobases) was measured and compared in two different human cell lines. In human erythroleukemia (K562) cells, in which embryonic and fetal globin genes are transcribed, all of the sequences we examined from the P-globin domain replicated early during S phase, while in HeLa cells, in which globin genes are transcriptionally silent, these sequences replicated late during S. Potential sites of initiation of DNA replication within this domain were identified. The 3-globin gene domain was also found to differ with respect to the nuclease sensitivity of the chromatin in these two cell lines. In K562 cells, hypersensitive sites for endogenous nucleases and DNase I were present in the chromatin near the earliest-replicating segments in the 13-globin domain.In viruses and procaryotes, DNA replication initiates at well-defined sequences (origins of replication) and proceeds bidirectionally. (IgCH) in mouse cell lines is also consistent with replication proceeding from a presumptive origin downstream of the gene cluster when the locus is inactive (5). However, when one or more genes in the IgCH cluster are transcribed, the entire cluster replicates at the same time at the beginning of S with no apparent temporal directionality (5). Thus, a change in the timing and possibly a change in the origin and direction of replication is associated with the change in the transcriptional activity within this cluster.To gain more information about the relationship between gene expression and the timing of DNA replication in mammalian cells, we have studied the human ,B-globin gene cluster. This gene cluster has been well characterized at the molecular level and consists of an embryonic (E) gene expressed in the early embryo, two fetal genes (Gy, AY) expressed in the fetal liver, two adult genes (8, I) expressed in adult bone marrow, and a pseudogene (i4. These genes undergo a switch in their expression during erythroid cell development. These genes are clustered in a 50-kilobase (kb) region on chromosome 11 and occur in the order 5'-Gyy8Ab-3' (11). Centrifugal elutriation was used to fractionate 5-bromodeoxyuridine (BUdR)-labeled human cells into various intervals of S phase, followed by detailed hybridization analysis of bromouracil (BU)-labeled, newly replicated DNA with DNA probes spanning the length of the human ,B-globin gene locus. This fractionation technique has a number of distinct advantages over other assays; it is more rapid, a large number of cells can be fractionated, and most importantly, it obviates the need for the toxic chemicals that are used in cell synchronization. In this report, we show that the timing of replication during the S phase of a contiguous stretch of chromosomal DNA (140 kb) containing the human 4958

The functionality of the tumor suppressor p53 is altered in more than 50% of human cancers, and many individuals with cancer exhibit amyloid-like buildups of aggregated p53. An understanding of what triggers the pathogenic amyloid conversion of p53 is required for the further development of cancer therapies. Here, perturbation of the p53 core domain (p53C) with subdenaturing concentrations of guanidine hydrochloride and high hydrostatic pressure revealed native-like molten globule (MG) states, a subset of which were highly prone to amyloidogenic aggregation. We found that MG conformers of p53C, probably representing population-weighted averages of multiple states, have different volumetric properties, as determined by pressure perturbation and size-exclusion chromatography. We also found that they bind the fluorescent dye 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid (bis-ANS) and have a native-like tertiary structure that occludes the single Trp residue in p53. Fluorescence experiments revealed conformational changes of the single Trp and Tyr residues before p53 unfolding and the presence of MG conformers, some of which were highly prone to aggregation. p53C exhibited marginal unfolding cooperativity, which could be modulated from unfolding to aggregation pathways with chemical or physical forces. We conclude that trapping amyloid precursor states in solution is a promising approach for understanding p53 aggregation in cancer. Our findings support the use of single-Trp fluorescence as a probe for evaluating p53 stability, effects of mutations, and the efficacy of therapeutics designed to stabilize p53.

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Edited by Roger J. ColbranHypertrophic cardiomyopathy (HCM) is one of the most common cardiomyopathies and a major cause of sudden death in young athletes. The Ca 2؉ sensor of the sarcomere, cardiac troponin C (cTnC), plays an important role in regulating muscle contraction. Although several cardiomyopathy-causing mutations have been identified in cTnC, the limited information about their structural defects has been mapped to the HCM phenotype. Here, we used high-resolution electron-spray ionization mass spectrometry (ESI-MS), Carr-Purcell-MeiboomGill relaxation dispersion (CPMG-RD), and affinity measurements of cTnC for the thin filament in reconstituted papillary muscles to provide evidence of an allosteric mechanism in mutant cTnC that may play a role to the HCM phenotype. We showed that the D145E mutation leads to altered dynamics on a s-ms time scale and deactivates both of the divalent cationbinding sites of the cTnC C-domain. CPMG-RD captured a low populated protein-folding conformation triggered by the Glu-145 replacement of Asp. Paradoxically, although D145E C-domain was unable to bind Ca 2؉ , these changes along its backbone allowed it to attach more firmly to thin filaments than the wildtype isoform, providing evidence for an allosteric response of the Ca 2؉ -binding site II in the N-domain. Our findings explain how the effects of an HCM mutation in the C-domain reflect up into the N-domain to cause an increase of Ca 2؉ affinity in site II, thus opening up new insights into the HCM phenotype.