U5 snRNA Interactions With Exons Ensure Splicing Precision.

Imperfect conservation of human pre-mRNA splice sites is necessary to produce alternative isoforms. This flexibility is combined with the precision of the message reading frame. Apart from intron-termini GU_AG and the branchpoint A, the most conserved are the exon-end guanine and +5G of the intron start. Association between these guanines cannot be explained solely by base-pairing with U1 snRNA in the early spliceosome complex. U6 succeeds U1 and pairs +5G in the pre-catalytic spliceosome, while U5 binds the exon end. Current U5 snRNA reconstructions by CryoEM cannot explain the conservation of the exon-end G. Conversely, human mutation analyses show that guanines of both exon termini can suppress splicing mutations. Our U5 hypothesis explains the mechanism of splicing precision and the role of these conserved guanines in the pre-catalytic spliceosome. We propose: (1) optimal binding register for human exons and U5-the exon junction positioned at U5Loop1 C39|C38; (2) common mechanism for base-pairing of human U5 snRNA with diverse exons and bacterial Ll.LtrB intron with new loci in retrotransposition-guided by base pair geometry; and (3) U5 plays a significant role in specific exon recognition in the pre-catalytic spliceosome. Statistical analyses showed increased U5 Watson-Crick pairs with the 5'exon in the absence of +5G at the intron start. In 5'exon positions -3 and -5, this effect is specific to U5 snRNA rather than U1 snRNA of the early spliceosome. Increased U5 Watson-Crick pairs with 3'exon position +1 coincide with substitutions of the conserved -3C at the intron 3'end. Based on mutation and X-ray evidence, we propose that -3C pairs with U2 G31 juxtaposing the branchpoint and the 3'intron end. The intron-termini pair, formed in the pre-catalytic spliceosome to be ready for transition after branching, and the early involvement of the 3'intron end ensure that the 3'exon contacts U5 in the pre-catalytic complex. We suggest that splicing precision is safeguarded cooperatively by U5, U6, and U2 snRNAs that stabilize the pre-catalytic complex by Watson-Crick base pairing. In addition, our new U5 model explains the splicing effect of exon-start +1G mutations: U5 Watson-Crick pairs with exon +2C/+3G strongly promote exon inclusion. We discuss potential applications for snRNA therapeutics and gene repair by reverse splicing.

Vulnerability of the North Water ecosystem to climate change.

High Arctic ecosystems and Indigenous livelihoods are tightly linked and exposed to climate change, yet assessing their sensitivity requires a long-term perspective. Here, we assess the vulnerability of the North Water polynya, a unique seaice ecosystem that sustains the world's northernmost Inuit communities and several keystone Arctic species. We reconstruct mid-to-late Holocene changes in sea ice, marine primary production, and little auk colony dynamics through multi-proxy analysis of marine and lake sediment cores. Our results suggest a productive ecosystem by 4400-4200 cal yrs b2k coincident with the arrival of the first humans in Greenland. Climate forcing during the late Holocene, leading to periods of polynya instability and marine productivity decline, is strikingly coeval with the human abandonment of Greenland from c. 2200-1200 cal yrs b2k. Our long-term perspective highlights the future decline of the North Water ecosystem, due to climate warming and changing sea-ice conditions, as an important climate change risk.

Coupled impacts of sea ice variability and North Pacific atmospheric circulation on Holocene hydroclimate in Arctic Alaska.

Arctic Alaska lies at a climatological crossroads between the Arctic and North Pacific Oceans. The modern hydroclimate of the region is responding to rapidly diminishing sea ice, driven in part by changes in heat flux from the North Pacific. Paleoclimate reconstructions have improved our knowledge of Alaska's hydroclimate, but no studies have examined Holocene sea ice, moisture, and ocean-atmosphere circulation in Arctic Alaska, limiting our understanding of the relationship between these phenomena in the past. Here we present a sedimentary diatom assemblage and diatom isotope dataset from Schrader Pond, located ∼80 km from the Arctic Ocean, which we interpret alongside synthesized regional records of Holocene hydroclimate and sea ice reduction scenarios modeled by the Hadley Centre Coupled Model Version 3 (HadCM3). The paleodata synthesis and model simulations suggest the Early and Middle Holocene in Arctic Alaska were characterized by less sea ice, a greater contribution of isotopically heavy Arctic-derived moisture, and wetter climate. In the Late Holocene, sea ice expanded and regional climate became drier. This climatic transition is coincident with a documented shift in North Pacific circulation involving the Aleutian Low at ∼4 ka, suggesting a Holocene teleconnection between the North Pacific and Arctic. The HadCM3 simulations reveal that reduced sea ice leads to a strengthened Aleutian Low shifted west, potentially increasing transport of warm North Pacific water to the Arctic through the Bering Strait. Our findings demonstrate the interconnectedness of the Arctic and North Pacific on multimillennial timescales, and are consistent with future projections of less sea ice and more precipitation in Arctic Alaska.

Human settlement of East Polynesia earlier, incremental, and coincident with prolonged South Pacific drought.

The timing of human colonization of East Polynesia, a vast area lying between Hawai'i, Rapa Nui, and New Zealand, is much debated and the underlying causes of this great migration have been enigmatic. Our study generates evidence for human dispersal into eastern Polynesia from islands to the west from around AD 900 and contemporaneous paleoclimate data from the likely source region. Lake cores from Atiu, Southern Cook Islands (SCIs) register evidence of pig and/or human occupation on a virgin landscape at this time, followed by changes in lake carbon around AD 1000 and significant anthropogenic disturbance from c. AD 1100. The broader paleoclimate context of these early voyages of exploration are derived from the Atiu lake core and complemented by additional lake cores from Samoa (directly west) and Vanuatu (southwest) and published hydroclimate proxies from the Society Islands (northeast) and Kiribati (north). Algal lipid and leaf wax biomarkers allow for comparisons of changing hydroclimate conditions across the region before, during, and after human arrival in the SCIs. The evidence indicates a prolonged drought in the likely western source region for these colonists, lasting c. 200 to 400 y, contemporaneous with the phasing of human dispersal into the Pacific. We propose that drying climate, coupled with documented social pressures and societal developments, instigated initial eastward exploration, resulting in SCI landfall(s) and return voyaging, with colonization a century or two later. This incremental settlement process likely involved the accumulation of critical maritime knowledge over several generations.

C mobilisation in disturbed tropical peat swamps: old DOC can fuel the fluvial efflux of old carbon dioxide, but site recovery can occur.

Southeast-Asian peat swamp forests have been significantly logged and converted to plantation. Recently, to mitigate land degradation and C losses, some areas have been left to regenerate. Understanding how such complex land use change affects greenhouse gas emissions is essential for modelling climate feedbacks and supporting land management decisions. We carried out field research in a Malaysian swamp forest and an oil palm plantation to understand how clear-felling, drainage, and illegal and authorized conversion to oil palm impacted the C cycle, and how the C cycle may change if such logging and conversion stopped. We found that both the swamp forest and the plantation emit centuries-old CO2 from their drainage systems in the managed areas, releasing sequestered C to the atmosphere. Oil palm plantations are an iconic symbol of tropical peatland degradation, but CO2 efflux from the recently-burnt, cleared swamp forest was as old as from the oil palm plantation. However, in the swamp forest site, where logging had ceased approximately 30 years ago, the age of the CO2 efflux was modern, indicating recovery of the system can occur. 14C dating of the C pool acted as a tracer of recovery as well as degradation and offers a new tool to assess efficacy of restoration management. Methane was present in many sites, and in higher concentrations in slow-flowing anoxic systems as degassing mechanisms are not strong. Methane loading in freshwaters is rarely considered, but this may be an important C pool in restored drainage channels and should be considered in C budgets and losses.

The Impact of the C-Terminal Region on the Interaction of Topoisomerase II Alpha with Mitotic Chromatin.

Type II topoisomerase enzymes are essential for resolving DNA topology problems arising through various aspects of DNA metabolism. In vertebrates two isoforms are present, one of which (TOP2A) accumulates on chromatin during mitosis. Moreover, TOP2A targets the mitotic centromere during prophase, persisting there until anaphase onset. It is the catalytically-dispensable C-terminal domain of TOP2 that is crucial in determining this isoform-specific behaviour. In this study we show that, in addition to the recently identified chromatin tether domain, several other features of the alpha-C-Terminal Domain (CTD). influence the mitotic localisation of TOP2A. Lysine 1240 is a major SUMOylation target in cycling human cells and the efficiency of this modification appears to be influenced by T1244 and S1247 phosphorylation. Replacement of K1240 by arginine results in fewer cells displaying centromeric TOP2A accumulation during prometaphase-metaphase. The same phenotype is displayed by cells expressing TOP2A in which either of the mitotic phosphorylation sites S1213 or S1247 has been substituted by alanine. Conversely, constitutive modification of TOP2A by fusion to SUMO2 exerts the opposite effect. FRAP analysis of protein mobility indicates that post-translational modification of TOP2A can influence the enzyme's residence time on mitotic chromatin, as well as its subcellular localisation.

Roles of the C-terminal domains of topoisomerase IIα and topoisomerase IIß in regulation of the decatenation checkpoint.

Topoisomerase (topo) IIα and IIß maintain genome stability and are targets for anti-tumor drugs. In this study, we demonstrate that the decatenation checkpoint is regulated, not only by topo IIα, as previously reported, but also by topo IIß. The decatenation checkpoint is most efficient when both isoforms are present. Regulation of this checkpoint and sensitivity to topo II-targeted drugs is influenced by the C-terminal domain (CTD) of the topo II isoforms and by a conserved non-catalytic tyrosine, Y640 in topo IIα and Y656 in topo IIß. Deletion of most of the CTD of topo IIα, while preserving the nuclear localization signal (NLS), enhances the decatenation checkpoint and sensitivity to topo II-targeted drugs. In contrast, deletion of most of the CTD of topo IIß, while preserving the NLS, and mutation of Y640 in topo IIα and Y656 in topo IIß inhibits these activities. Structural studies suggest that the differential impact of the CTD on topo IIα and topo IIß function may be due to differences in CTD charge distribution and differential alignment of the CTD with reference to transport DNA. Together these results suggest that topo IIα and topo IIß cooperate to maintain genome stability, which may be distinctly modulated by their CTDs.

Pregnancy termination for fetal abnormality: are health professionals' perceptions of women's coping congruent with women's accounts?

BACKGROUND: Pregnancy termination for fetal abnormality (TFA) may have profound psychological consequences for those involved. Evidence suggests that women's experience of care influences their psychological adjustment to TFA and that they greatly value compassionate healthcare. Caring for women in these circumstances presents challenges for health professionals, which may relate to their understanding of women's experience. This qualitative study examined health professionals' perceptions of women's coping with TFA and assessed to what extent these perceptions are congruent with women's accounts. METHODS: Fifteen semi-structured interviews were carried out with health professionals in three hospitals in England. Data were analysed using thematic analysis and compared with women's accounts of their own coping processes to identify similarities and differences. RESULTS: Health professionals' perceptions of women's coping processes were congruent with women's accounts in identifying the roles of support, acceptance, problem-solving, avoidance, another pregnancy and meaning attribution as key coping strategies. Health professionals regarded coping with TFA as a unique grieving process and were cognisant of women's idiosyncrasies in coping. They also considered their role as information providers as essential in helping women cope with TFA. The findings also indicate that health professionals lacked insight into women's long-term coping processes and the potential for positive growth following TFA, which is consistent with a lack of aftercare following TFA reported by women. CONCLUSIONS: Health professionals' perceptions of women's coping with TFA closely matched women's accounts, suggesting a high level of understanding. However, the lack of insight into women's long-term coping processes has important clinical implications, as research suggests that coping with TFA is a long-term process and that the provision of aftercare is beneficial to women. Together, these findings call for further research into the most appropriate ways to support women post-TFA, with a view to developing a psychological intervention to better support women in the future.

Synthetic Lethal Screen Demonstrates That a JAK2 Inhibitor Suppresses a BCL6-dependent IL10RA/JAK2/STAT3 Pathway in High Grade B-cell Lymphoma.

We demonstrate the usefulness of synthetic lethal screening of a conditionally BCL6-deficient Burkitt lymphoma cell line, DG75-AB7, with a library of small molecules to determine survival pathways suppressed by BCL6 and suggest mechanism-based treatments for lymphoma. Lestaurtinib, a JAK2 inhibitor and one of the hits from the screen, repressed survival of BCL6-deficient cells in vitro and reduced growth and proliferation of xenografts in vivo BCL6 deficiency in DG75-AB7 induced JAK2 mRNA and protein expression and STAT3 phosphorylation. Surface IL10RA was elevated by BCL6 deficiency, and blockade of IL10RA repressed STAT3 phosphorylation. Therefore, we define an IL10RA/JAK2/STAT3 pathway each component of which is repressed by BCL6. We also show for the first time that JAK2 is a direct BCL6 target gene; BCL6 bound to the JAK2 promoter in vitro and was enriched by ChIP-seq. The place of JAK2 inhibitors in the treatment of diffuse large B-cell lymphoma has not been defined; we suggest that JAK2 inhibitors might be most effective in poor prognosis ABC-DLBCL, which shows higher levels of IL10RA, JAK2, and STAT3 but lower levels of BCL6 than GC-DLBCL and might be usefully combined with novel approaches such as inhibition of IL10RA.

A role for human homologous recombination factors in suppressing microhomology-mediated end joining.

DNA double-strand breaks (DSBs) are toxic lesions, which if improperly repaired can result in cell death or genomic instability. DSB repair is usually facilitated by the classical non-homologous end joining (C-NHEJ), or homologous recombination (HR) pathways. However, a mutagenic alternative NHEJ pathway, microhomology-mediated end joining (MMEJ), can also be deployed. While MMEJ is suppressed by C-NHEJ, the relationship between HR and MMEJ is less clear. Here, we describe a role for HR genes in suppressing MMEJ in human cells. By monitoring DSB mis-repair using a sensitive HPRT assay, we found that depletion of HR proteins, including BRCA2, BRCA1 or RPA, resulted in a distinct mutational signature associated with significant increases in break-induced mutation frequencies, deletion lengths and the annealing of short regions of microhomology (2-6 bp) across the break-site. This signature was dependent on CtIP, MRE11, POLQ and PARP, and thus indicative of MMEJ. In contrast to CtIP or MRE11, depletion of BRCA1 resulted in increased partial resection and MMEJ, thus revealing a functional distinction between these early acting HR factors. Together these findings indicate that HR factors suppress mutagenic MMEJ following DSB resection.

Carbon isotope discrimination in leaves of the broad-leaved paperbark tree, Melaleuca quinquenervia, as a tool for quantifying past tropical and subtropical rainfall.

Quantitative reconstructions of terrestrial climate are highly sought after but rare, particularly in Australia. Carbon isotope discrimination in plant leaves (Δleaf ) is an established indicator of past hydroclimate because the fractionation of carbon isotopes during photosynthesis is strongly influenced by water stress. Leaves of the evergreen tree Melaleuca quinquenervia have been recovered from the sediments of some perched lakes on North Stradbroke and Fraser Islands, south-east Queensland, eastern Australia. Here, we examine the potential for using M. quinquenervia ∆leaf as a tracer of past rainfall by analysing carbon isotope ratios (δ(13) C) of modern leaves. We firstly assess Δleaf variation at the leaf and stand scale and find no systematic pattern within leaves or between leaves due to their position on the tree. We then examine the relationships between climate and Δleaf for a 11-year time series of leaves collected in a litter tray. M. quinquenervia retains its leaves for 1-4 years; thus, cumulative average climate data are used. There is a significant relationship between annual mean ∆leaf and mean annual rainfall of the hydrological year for 1-4 years (i.e. 365-1460 days) prior to leaf fall (r(2)  = 0.64, P = 0.003, n = 11). This relationship is marginally improved by accounting for the effect of pCO2 on discrimination (r(2)  = 0.67, P = 0.002, n = 11). The correlation between rainfall and Δleaf , and the natural distribution of Melaleuca quinquenervia around wetlands of eastern Australia, Papua New Guinea and New Caledonia offers significant potential to infer past rainfall on a wide range of spatial and temporal scales.

Use of the HPRT gene to study nuclease-induced DNA double-strand break repair.

Understanding the mechanisms of chromosomal double-strand break repair (DSBR) provides insight into genome instability, oncogenesis and genome engineering, including disease gene correction. Research into DSBR exploits rare-cutting endonucleases to cleave exogenous reporter constructs integrated into the genome. Multiple reporter constructs have been developed to detect various DSBR pathways. Here, using a single endogenous reporter gene, the X-chromosomal disease gene encoding hypoxanthine phosphoribosyltransferase (HPRT), we monitor the relative utilization of three DSBR pathways following cleavage by I-SceI or CRISPR/Cas9 nucleases. For I-SceI, our estimated frequencies of accurate or mutagenic non-homologous end-joining and gene correction by homologous recombination are 4.1, 1.5 and 0.16%, respectively. Unexpectedly, I-SceI and Cas9 induced markedly different DSBR profiles. Also, using an I-SceI-sensitive HPRT minigene, we show that gene correction is more efficient when using long double-stranded DNA than single- or double-stranded oligonucleotides. Finally, using both endogenous HPRT and exogenous reporters, we validate novel cell cycle phase-specific I-SceI derivatives for investigating cell cycle variations in DSBR. The results obtained using these novel approaches provide new insights into template design for gene correction and the relationships between multiple DSBR pathways at a single endogenous disease gene.

Stimulation of oligonucleotide-directed gene correction by Redß expression and MSH2 depletion in human HT1080 cells.

The correction of disease-causing mutations by single-strand oligonucleotide-templated DNA repair (ssOR) is an attractive approach to gene therapy, but major improvements in ssOR efficiency and consistency are needed. The mechanism of ssOR is poorly understood but may involve annealing of oligonucleotides to transiently exposed single-stranded regions in the target duplex. In bacteria and yeast it has been shown that ssOR is promoted by expression of Redß, a single-strand DNA annealing protein from bacteriophage lambda. Here we show that Redß expression is well tolerated in a human cell line where it consistently promotes ssOR. By use of short interfering RNA, we also show that ssOR is stimulated by the transient depletion of the endogenous DNA mismatch repair protein MSH2. Furthermore, we find that the effects of Redß expression and MSH2 depletion on ssOR can be combined with a degree of cooperativity. These results suggest that oligonucleotide annealing and mismatch recognition are distinct but interdependent events in ssOR that can be usefully modulated in gene correction strategies.

SETD2-dependent histone H3K36 trimethylation is required for homologous recombination repair and genome stability.

Modulating chromatin through histone methylation orchestrates numerous cellular processes. SETD2-dependent trimethylation of histone H3K36 is associated with active transcription. Here, we define a role for H3K36 trimethylation in homologous recombination (HR) repair in human cells. We find that depleting SETD2 generates a mutation signature resembling RAD51 depletion at I-SceI-induced DNA double-strand break (DSB) sites, with significantly increased deletions arising through microhomology-mediated end-joining. We establish a presynaptic role for SETD2 methyltransferase in HR, where it facilitates the recruitment of C-terminal binding protein interacting protein (CtIP) and promotes DSB resection, allowing Replication Protein A (RPA) and RAD51 binding to DNA damage sites. Furthermore, reducing H3K36me3 levels by overexpressing KDM4A/JMJD2A, an oncogene and H3K36me3/2 demethylase, or an H3.3K36M transgene also reduces HR repair events. We propose that error-free HR repair within H3K36me3-decorated transcriptionally active genomic regions promotes cell homeostasis. Moreover, these findings provide insights as to why oncogenic mutations cluster within the H3K36me3 axis.

Nuclease-stimulated homologous recombination at the human ß-globin gene.

BACKGROUND: Mutations in the ß-globin gene (HBB) cause haemoglobinopathies where current treatments have serious limitations. Gene correction by homologous recombination (HR) is an attractive approach to gene therapy for such diseases and is stimulated by gene-specific endonucleases, including zinc finger nucleases (ZFNs). Customised nucleases targeting HBB have previously been shown to promote HR-mediated HBB modification in 0.3­60% of drug-selected cells, although frequencies among unselected cells, more relevant to the goal of correcting HBB in primary stem cells, have not been reported. METHODS: ZFNs targeting HBB were tested for HBB binding (two-hybrid assay) or HBB cleavage followed by inaccurate end joining (surveyor assay)in bacteria or human cancer cell lines, respectively. ZFN-stimulated HR was measured in cell lines by a modified fluorescence-based reporter assay or by targeted insertion of a drug-resistance marker into endogenous HBB confirmed by Southern analyses. RESULTS: Although the ZFNs that we assembled in-house showed limited potential, a commercially commissioned nuclease (ZFN4) enhanced HR mediated HBB modification in up to 95% of drug-selected cells. Among unselected cells, however, this frequency was less than 0.2%. Furthermore, ZFN4 cleaved HBB at an efficiency of 1­2% (surveyor assay) and enhanced the HR reporter assay 20-fold less efficiently than a control endonuclease. CONCLUSIONS: With ZFN4, we achieved higher efficiencies of HR-mediated HBB modification than previously reported for drug-selected cells. Our measurements of ZFN4-induced HR in unselected cells, however, suggest that improved nucleases must be developed if therapeutic HBB correction is to be achievable in primary stem cells.

The α isoform of topoisomerase II is required for hypercompaction of mitotic chromosomes in human cells.

As proliferating cells transit from interphase into M-phase, chromatin undergoes extensive reorganization, and topoisomerase (topo) IIα, the major isoform of this enzyme present in cycling vertebrate cells, plays a key role in this process. In this study, a human cell line conditional null mutant for topo IIα and a derivative expressing an auxin-inducible degron (AID)-tagged version of the protein have been used to distinguish real mitotic chromosome functions of topo IIα from its more general role in DNA metabolism and to investigate whether topo IIß makes any contribution to mitotic chromosome formation. We show that topo IIß does contribute, with endogenous levels being sufficient for the initial stages of axial shortening. However, a significant effect of topo IIα depletion, seen with or without the co-depletion of topo IIß, is the failure of chromosomes to hypercompact when delayed in M-phase. This requires much higher levels of topo II protein and is impaired by drugs or mutations that affect enzyme activity. A prolonged delay at the G2/M border results in hyperefficient axial shortening, a process that is topo IIα-dependent. Rapid depletion of topo IIα has allowed us to show that its function during late G2 and M-phase is truly required for shaping mitotic chromosomes.

Treatment with gefitinib or lapatinib induces drug resistance through downregulation of topoisomerase IIα expression.

The EGF receptor (EGFR) is therapeutically targeted by antibodies and small molecules in solid tumors including lung, colorectal, and breast cancer. However, chemotherapy remains important, and efforts to improve efficacy through combination with targeted agents is challenging. This study examined the effects of short and long durations of exposure to the EGFR- and HER2-targeted tyrosine kinase inhibitors (TKI) gefitinib and lapatinib, on induction of cell death and DNA damage by topoisomerase IIα (Topo IIα) poisons, in the SK-Br-3 HER2-amplified breast cancer cell line. Short exposure to either gefitinib or lapatinib for 1 hour did not affect the induction of apoptosis by the Topo IIα poisons doxorubicin, etoposide, and m-AMSA. In contrast, cells treated for 48 hours were resistant to all three drugs. Short exposure (1 hour) to TKI did not alter the number of DNA single- or double-strand breaks (DSB) induced, whereas longer exposure (48 hours) reduced the number of DNA DSBs and the formation of γ-H2AX foci. Both gefitinib and lapatinib reduced the expression and activity of Topo IIα at 48 hours. Studies using a cell line with inducible downregulation of Topo IIα showed that expression of Topo IIα, and not Topo IIß, determined the number of DNA strand breaks induced by these chemotherapeutic agents. These results indicate that prolonged exposure to TKIs targeting EGFR and HER2 induce resistance to doxorubicin, etoposide, and m-AMSA through downregulation of Topo IIα. This may explain why their addition to chemotherapy regimens have not increased efficacy.

Topoisomerase IIα promotes activation of RNA polymerase I transcription by facilitating pre-initiation complex formation.

Type II DNA topoisomerases catalyse DNA double-strand cleavage, passage and re-ligation to effect topological changes. There is considerable interest in elucidating topoisomerase II roles, particularly as these proteins are targets for anti-cancer drugs. Here we uncover a role for topoisomerase IIα in RNA polymerase I-directed ribosomal RNA gene transcription, which drives cell growth and proliferation and is upregulated in cancer cells. Our data suggest that topoisomerase IIα is a component of the initiation-competent RNA polymerase Iß complex and interacts directly with RNA polymerase I-associated transcription factor RRN3, which targets the polymerase to promoter-bound SL1 in pre-initiation complex formation. In cells, activation of rDNA transcription is reduced by inhibition or depletion of topoisomerase II, and this is accompanied by reduced transient double-strand DNA cleavage in the rDNA-promoter region and reduced pre-initiation complex formation. We propose that topoisomerase IIα functions in RNA polymerase I transcription to produce topological changes at the rDNA promoter that facilitate efficient de novo pre-initiation complex formation.

Chemical genetic analyses of quantitative changes in Cdk1 activity during the human cell cycle.

Cyclin-dependent kinase 1 (Cdk1) controls cell proliferation and is inhibited by promising anticancer agents, but its mode of action and the consequences of its inhibition are incompletely understood. Cdk1 promotes S- and M-phases during the cell-cycle but also suppresses endoreduplication, which is associated with polyploidy and genome instability. The complexity of Cdk1 regulation has made it difficult to determine whether these different roles require different thresholds of kinase activity and whether the surge of activity as inhibitory phosphates are removed at mitotic onset is essential for cell proliferation. Here, we have used chemical genetics in a human cell line to address these issues. We rescued cells lethally depleted of endogenous Cdk1 with an exogenous Cdk1 conferring sensitivity to one ATP analogue inhibitor (1NMPP1) and resistance to another (RO3306). At no 1NMPP1 concentration was mitosis in rescued clones prevented without also inducing endoreduplication, suggesting that these two key roles for Cdk1 are not simply controlled by different Cdk1 activity thresholds. We also rescued RO3306-resistant clones using exogenous Cdk1 without inhibitory phosphorylation sites, indicating that the mitotic surge of Cdk1 activity is dispensable for cell proliferation. These results suggest that the basic mammalian cycle requires at least some qualitative changes in Cdk1 activity and that quantitative increases in activity need not be rapid. Furthermore, the viability of cells that are unable to undergo rapid Cdk1 activation, and the strong association between endoreduplication and impaired proliferation, may place restrictions on the therapeutic use of a Cdk1 inhibitors.

Ataxia telangiectasia mutated-dependent regulation of topoisomerase II alpha expression and sensitivity to topoisomerase II inhibitor.

Topoisomerase II alpha (TOP2A) has a crucial role in proper chromosome condensation and segregation. Here we report the interaction of TOP2A with ataxia telangiectasia mutated (ATM) and its phosphorylation in an ATM-dependent manner after DNA damage. In vitro kinase assay and site-directed mutagenesis studies revealed that serine 1512 is the target of phosphorylation through ATM. Serine 1512 to Alanine mutation of TOP2A showed increased stability of the protein, retaining TOP2A activity at least with regard to cell survival activity. Ataxia telangiectasia-derived cell lines showed high levels of TOP2A that were associated with hypersensitivity to the TOP2 inhibitor etoposide. These findings suggest that ATM-dependent TOP2A modification is required for proper regulation of TOP2 stability and subsequently of the sensitivity to TOP2 inhibitor. In a lymphoblastoid cell line derived from a patient who developed MLL rearrangement, positive infant leukemia, defective ATM expression, and increased TOP2A expression were shown. It was intriguing that hypersensitivity to TOP2 inhibitor and susceptibility to MLL gene rearrangement were shown by low-dose etoposide exposure in this cell line. Thus, our findings have clinically important implications for the pathogenesis of infantile acute leukemia as well as treatment-associated secondary leukemia following exposure to TOP2 inhibitors.