We have previously reported the mobility of positioned nucleosomes on sea urchin 5S rDNA. In this study we demonstrate the temperature dependence and the range of this mobility on 5S rDNA constructs. We find that this dynamic behavior also applies to bulk mononucleosomes and nucleosomes reconstituted onto sequences of the Alu family of ubiquitous repeats. We conclude that short range sliding is potentially a general phenomenon that is dependent on the underlying sequence and its position on the histone octamer. The nucleoprotein gel analysis used also reveals the dramatic effect on gel electrophoretic migration caused by the location of the histone octamer on DNA fragments. The usefulness of this technique for studying nucleosome positioning and its dynamics is demonstrated.

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We have previously identified a generally occurring short-range mobility of nucleosome cores on DNA in relatively low ionic strength conditions. Here we report that this mobility of histone octamers positioned on constructs of 5S rDNA is suppressed by the binding of histone Hi or H5 to the nuceosome. Histone HS Is the more potent inhibitor of nudeosome mobility, in accordance with its higher affinity for chromatin. We propose that this reversible restraint on chromatin dynamics may play a role in local regulation of processes that require access to the DNA. H1 has been identified as a general repressor of transcription (6, 7). It was assumed previously that active genes were devoid of H1 and that Hl-induced higher-order structures caused repression. Recent studies, however, show that H1 remains in active chromatin fractions, probably in reduced amounts or with aweakened afflnity (8)(9)(10)(11). This modified association ofHi with active chromatin may allow access to the DNA.It should be noted that even without H1, the packaging of DNA into nucleosome cores in itselfrenders a large component of the DNA sequences inaccessible to trans-acting factors (for reviews, see refs. 12 and 13). The mechanisms of eukaryotic DNA processing probably involve a dynamic behavior of the nucleosome structure. Various modes of nucleosome disruption, nucleosome transfer, and histone dissociation have been invoked in models for initiation and elongation of transcription (for reviews, see refs. 13 and 14). We have identified (iS) a general mobility of nucleosomes under conditions of relatively low ionic strength that may be relevant to these models. Nucleosome cores containing the full histone octamer exhibit short-range mobility over 1O-bp DNA intervals. Thus the rotational setting of the DNA around the octamer is conserved during these nucleosome core movements (16). This temperature-dependent short-range mobility is distinct from the previously observed nucleosome sliding at relatively high ionic strengths (17, 18), which probably results from the weakening of histone-DNA interactions.Because of the probable importance of H1 as a general repressor, we have investigated the effect of the binding of linker histones on nucleosome mobility. Nucleosome positioning on sea urchin 58 rDNA is well characterized both without (19)(20)(21) and with (21) bound linker histones. We show that histones H1 and H5 (a member of the H1 family found in nucleated erythrocytes) effectively suppress the redistribution of histone octamers between possible nucleosome positions on this DNA. If nucleosome mobility is required for access to the DNA, then H1 could function as a repressor outside the context of the 30-nm fiber through its immobilization of nucleosome cores. MATERIALS AND METHODSPreparation of DNA Substrates. The 207-bp sea urchin 5S rDNA fragments were generated from the tandemly repeated insert of plasmid p5S207-18 (22) by Ava I restriction digestion. Head-to-tail dimers of this sequence were obtained by ligation at the asymmetric Ava I s...

BackgroundBRAF V600 mutant circulating cell-free tumor DNA (BRAF V600mut ctDNA) could serve as a specific biomarker in patients with BRAF V600 mutant melanoma. We analyzed the value of BRAF V600mut ctDNA from plasma as a monitoring tool for advanced melanoma patients treated with BRAF/MEK inhibitors.MethodsAllele-specific quantitative PCR analysis for BRAF V600 E/E2/D/K/R/M mutations was performed on DNA extracted from plasma of patients with known BRAF V600 mutant melanoma who were treated with dabrafenib and trametinib.Results245 plasma samples from 36 patients were analyzed. In 16 patients the first plasma sample was obtained before the first dosing of dabrafenib/trametinib. At baseline, BRAF V600mut ctDNA was detected in 75 % of patients (n = 12/16). BRAF V600mut ctDNA decreased rapidly upon initiation of targeted therapy (p < 0.001) and became undetectable in 60 % of patients (n = 7/12) after 6 weeks of treatment. During treatment, disease progression (PD) was diagnosed in 27 of 36 patients. An increase of the BRAF V600mut ctDNA copy number and fraction, identified PD with a sensitivity of 70 % (n = 19/27) and a specificity of 100 %. An increase in the BRAF V600mut ctDNA fraction was detected prior to clinical PD in 44 % of cases (n = 12/27) and simultaneously with PD in 26 % of patients (n = 7/27).ConclusionsQuantitative analysis of BRAF V600mut ctDNA in plasma has unique features as a monitoring tool during treatment with BRAF/MEK inhibitors. Its potential as an early predictor of acquired resistance deserves further evaluation.

Integration of viral DNA into the host chromosome is an essential step in the life cycle of retroviruses and is facilitated by the viral integrase enzyme. The first generation of integrase inhibitors recently approved or currently in late-stage clinical trials shows great promise for the treatment of human immunodeficiency virus (HIV) infection, but virus is expected to develop resistance to these drugs. Therefore, we used a novel resistance selection protocol to follow the emergence of resistant HIV in the presence of the integrase inhibitor elvitegravir (GS-9137). We find the primary resistance-conferring mutations to be Q148R, E92Q, and T66I and demonstrate that they confer a reduction in susceptibility not only to elvitegravir but also to raltegravir (MK-0518) and other integrase inhibitors. The locations of the mutations are highlighted in the catalytic sites of integrase, and we correlate the mutations with expected drug-protein contacts. In addition, mutations that do not confer reduced susceptibility when present alone (H114Y, L74M, R20K, A128T, E138K, and S230R) are also discussed in relation to their position in the catalytic core domain and their proximity to known structural features of integrase. These data broaden the understanding of antiviral resistance against integrase inhibitors and may give insight facilitating the discovery of second-generation compounds.Integration of retroviral DNA is an essential step in the life cycle of human immunodeficiency virus (HIV) (29). The integration process is facilitated by the viral integrase (IN) enzyme which catalyzes the insertion of the viral DNA into the host genome in a multistep process involving viral and host proteins. HIV IN recognizes and binds specific sequences in the long terminal repeats (LTRs) of the viral retrotranscribed DNA in the cytoplasm. After DNA binding, IN cleaves GT dinucleotides from the 3Ј termini of the linear cDNA in a process called 3Ј processing (2). The processed viral DNA, as part of the preintegration complex, is then translocated into the nucleus, where IN inserts the viral DNA into the host chromosome by a process called strand transfer (2,12,13). There are few cellular enzymes with comparable function to HIV integrase (24), apart from the V(D)J polynucleotide recombinase RAG1 (34). Therefore, the IN enzyme has been considered an attractive target for antiretroviral therapy for the last decade (27, 36). Recent progress has resulted in two IN inhibitors (5, 30), with one drug in late-stage clinical trials and one currently approved for use in treatment-experienced patients (18). For all currently targeted retroviral proteins, inhibition with antiretroviral drugs has led to emergence of resistance in treated patients, often leading to treatment failure and requiring changes in the composition of the highly active antiretroviral therapy (HAART) drug regimen (6). Nevertheless, the emergence of new classes of drugs will enable new combinations of inhibitors to be used and will offer more treatment options to HIV-infected pati...