Single genome retrieval of context-dependent variability in mutation rates for human germline

Abstract Background Accurate knowledge of the core components of substitution rates is of vital importance to understand genome evolution and dynamics. By performing a single-genome and direct analysis of 39,894 retrotransposon remnants, we reveal sequence context-dependent germline nucleotide substitution rates for the human genome. Results The rates are characterised through rate constants in a time-domain, and are made available through a dedicated program (Trek) and a stand-alone database. Due to the nature of the method design and the imposed stringency criteria, we expect our rate constants to be good estimates for the rates of spontaneous mutations. Benefiting from such data, we study the short-range nucleotide (up to 7-mer) organisation and the germline basal substitution propensity (BSP) profile of the human genome; characterise novel, CpG-independent, substitution prone and resistant motifs; confirm a decreased tendency of moieties with low BSP to undergo somatic mutations in a number of cancer types; and, produce a Trek-based estimate of the overall mutation rate in human. Conclusions The extended set of rate constants we report may enrich our resources and help advance our understanding of genome dynamics and evolution, with possible implications for the role of spontaneous mutations in the emergence of pathological genotypes and neutral evolution of proteomes

Long genes and genes with multiple splice variants are enriched in pathways linked to cancer and other multigenic diseases

Background The role of random mutations and genetic errors in defining the etiology of cancer and other multigenic diseases has recently received much attention. With the view that complex genes should be particularly vulnerable to such events, here we explore the link between the simple properties of the human genes, such as transcript length, number of splice variants, exon/intron composition, and their involvement in the pathways linked to cancer and other multigenic diseases. Results We reveal a substantial enrichment of cancer pathways with long genes and genes that have multiple splice variants. Although the latter two factors are interdependent, we show that the overall gene length and splicing complexity increase in cancer pathways in a partially decoupled manner. Our systematic survey for the pathways enriched with top lengthy genes and with genes that have multiple splice variants reveal, along with cancer pathways, the pathways involved in various neuronal processes, cardiomyopathies and type II diabetes. We outline a correlation between the gene length and the number of somatic mutations. Conclusions Our work is a step forward in the assessment of the role of simple gene characteristics in cancer and a wider range of multigenic diseases. We demonstrate a significant accumulation of long genes and genes with multiple splice variants in pathways of multigenic diseases that have already been associated with de novo mutations. Unlike the cancer pathways, we note that the pathways of neuronal processes, cardiomyopathies and type II diabetes contain genes long enough for topoisomerase-dependent gene expression to also be a potential contributing factor in the emergence of pathologies, should topoisomerases become impaired.This research was supported by the Cancer Research UK and the Herchel Smith Fund. SB is a Wellcome Trust Senior Investigator.This is the final version of the article. It first appeared from BioMed Central via http://dx.doi.org/10.1186/s12864-016-2582-

Targeted Detection of G-Quadruplexes in Cellular RNAs.

The G-quadruplex (G4) is a non-canonical nucleic acid structure which regulates important cellular processes. RNA G4s have recently been shown to exist in human cells and be biologically significant. Described herein is a new approach to detect and map RNA G4s in cellular transcripts. This method exploits the specific control of RNA G4-cation and RNA G4-ligand interactions during reverse transcription, by using a selective reverse transcriptase to monitor RNA G4-mediated reverse transcriptase stalling (RTS) events. Importantly, a ligation-amplification strategy is coupled with RTS, and enables detection and mapping of G4s in important, low-abundance cellular RNAs. Strong evidence is provided for G4 formation in full-length cellular human telomerase RNA, offering important insights into its cellular function.This study is supported by a European Research Council Advanced grant to S.B. and supports C.K.K., and the Croucher Foundation for a fellowship to C.K.K. We thank Dr. M. Di Antonio, V. Chambers, and G. Mclnroy for providing comments on the manuscript.This is the final version of the article. It first appeared from Wiley via http://dx.doi.org/10.1002/anie.20150089

Reprogramming the mechanism of action of chlorambucil by coupling to a G-quadruplex ligand.

The nitrogen mustard Chlorambucil (Chl) generates covalent adducts with double-helical DNA and inhibits cell proliferation. Among these adducts, interstrand cross-links (ICLs) are the most toxic, as they stall replication by generating DNA double strand breaks (DSBs). Conversely, intrastrand cross-links generated by Chl are efficiently repaired by a dedicated Nucleotide Excision Repair (NER) enzyme. We synthesized a novel cross-linking agent that combines Chl with the G-quadruplex (G4) ligand PDS (PDS-Chl). We demonstrated that PDS-Chl alkylates G4 structures at low μM concentrations, without reactivity toward double- or single-stranded DNA. Since intramolecular G4s arise from a single DNA strand, we reasoned that preferential alkylation of such structures might prevent the generation of ICLs, while favoring intrastrand cross-links. We observed that PDS-Chl selectively impairs growth in cells genetically deficient in NER, but did not show any sensitivity to the repair gene BRCA2, involved in double-stranded break repair. Our findings suggest that G4 targeting of this clinically important alkylating agent alters the overall mechanism of action. These insights may inspire new opportunities for intervention in diseases specifically characterized by genetic impairment of NER, such as skin and testicular cancers.This is the final published version. It was originally published here: http://pubs.acs.org/doi/abs/10.1021/ja5014344

Bang-bang shortcut to adiabaticity in trapped-ion quantum simulators

We model the bang-bang optimization protocol as a shortcut to adiabaticity in the ground-state preparation of a trapped-ion quantum simulator. Compared to a locally adiabatic evolution, the bang-bang protocol typically produces a lower ground-state probability, but its implementation is so much simpler than the locally adiabatic approach, that it can become a competitive choice to use for maximizing ground-state preparation in systems that cannot be solved with conventional computers. We describe how one can optimize the shortcut and provide specific details for how it can be implemented with current trapped-ion quantum simulators. However, when frustration is strong enough, no method appears to work well for adiabatic state preparation within the experimental time frames, and one must confront the issue of dealing with diabatic excitations within the simulation.National Science Foundation (U.S.) (Grant PHY-1314295)National Science Foundation (U.S.) (Grant PHY-1620555

Selective Chemical Labeling of Natural T Modifications in DNA.

We present a chemical method to selectively tag and enrich thymine modifications, 5-formyluracil (5-fU) and 5-hydroxymethyluracil (5-hmU), found naturally in DNA. Inherent reactivity differences have enabled us to tag 5-fU chemoselectively over its C modification counterpart, 5-formylcytosine (5-fC). We rationalized the enhanced reactivity of 5-fU compared to 5-fC via ab initio quantum mechanical calculations. We exploited this chemical tagging reaction to provide proof of concept for the enrichment of 5-fU containing DNA from a pool that contains 5-fC or no modification. We further demonstrate that 5-hmU can be chemically oxidized to 5-fU, providing a strategy for the enrichment of 5-hmU. These methods will enable the mapping of 5-fU and 5-hmU in genomic DNA, to provide insights into their functional role and dynamics in biology.R.E.H. is supported by The University of Cambridge, F.K. is supported by the Wellcome Trust, and A.B.S. is supported by the Herchel Smith Fund. The Balasubramanian group is core- funded by a Wellcome Trust Senior Investigator Award and by Cancer Research UK. Departmental NMR facilities are supported by EPSRC grant EP/K039520/1.This is the final version. It was first published by ACS at http://pubs.acs.org/doi/abs/10.1021/jacs.5b03730

Dual binding of an antibody and a small molecule increases the stability of TERRA G-quadruplex.

In investigating the binding interactions between the human telomeric RNA (TERRA) G-quadruplex (GQ) and its ligands, it was found that the small molecule carboxypyridostatin (cPDS) and the GQ-selective antibody BG4 simultaneously bind the TERRA GQ. We previously showed that the overall binding affinity of BG4 for RNA GQs is not significantly affected in the presence of cPDS. However, single-molecule mechanical unfolding experiments revealed a population (48%) with substantially increased mechanical and thermodynamic stability. Force-jump kinetic investigations suggested competitive binding of cPDS and BG4 to the TERRA GQ. Following this, the two bound ligands slowly rearrange, thereby leading to the minor population with increased stability. Given the relevance of G-quadruplexes in the regulation of biological processes, we anticipate that the unprecedented conformational rearrangement observed in the TERRA-GQ-ligand complex may inspire new strategies for the selective stabilization of G-quadruplexes in cells.H.M. acknowledges support from NSF CHE-1026532. The Balasubramanian lab is supported by programme funding from Cancer Research UK.This is the final published version. It first appeared at http://onlinelibrary.wiley.com/doi/10.1002/anie.201408113/abstract;jsessionid=BB18FC03F2AF0C3EB95EC57CCBDB3DB9.f01t01

Accurate measurement of 5-methylcytosine and 5-hydroxymethylcytosine in human cerebellum DNA by oxidative bisulfite on an array (OxBS-array).

The Infinium 450K Methylation array is an established tool for measuring methylation. However, the bisulfite (BS) reaction commonly used with the 450K array cannot distinguish between 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). The oxidative-bisulfite assay disambiguates 5mC and 5hmC. We describe the use of oxBS in conjunction with the 450K array (oxBS-array) to analyse 5hmC/5mC in cerebellum DNA. The "methylation" level derived by the BS reaction is the combined level of 5mC and 5hmC at a given base, while the oxBS reaction gives the level of 5mC alone. The level of 5hmC is derived by subtracting the oxBS level from the BS level. Here we present an analysis method that distinguishes genuine positive levels of 5hmC at levels as low as 3%. We performed four replicates of the same sample of cerebellum and found a high level of reproducibility (average r for BS = 98.3, and average r for oxBS = 96.8). In total, 114,734 probes showed a significant positive measurement for 5hmC. The range at which we were able to distinguish 5hmC occupancy was between 3% and 42%. In order to investigate the effects of multiple replicates on 5hmC detection we also simulated fewer replicates and found that decreasing the number of replicates to two reduced the number of positive probes identified by > 50%. We validated our results using qPCR in conjunction with glucosylation of 5hmC sites followed by MspI digestion and we found good concordance with the array estimates (r = 0.94). This experiment provides a map of 5hmC in the cerebellum and a robust dataset for use as a standard in future 5hmC analyses. We also provide a novel method for validating the presence of 5hmC at low levels, and highlight some of the pitfalls associated with measuring 5hmC and 5mC.S. Balasubramanian is a Senior Investigator of The Wellcome Trust and the Balasubramanian group is core-funded by Cancer Research UK. We would like to thank Tobias Ost and Christine Clark of Cambridge Epigenetix Ltd. for valuable discussions and development of the method.This article was originally published in PLOS ONE (Field SF, Beraldi D, Bachman M, Stewart SK, Beck S, Balasubramanian S, PLoS ONE 2015, 10(2): e0118202. doi:10.1371/journal.pone.0118202

An Epigenetics-Inspired DNA-Based Data Storage System.

Biopolymers are an attractive alternative to store and circulate information. DNA, for example, combines remarkable longevity with high data storage densities and has been demonstrated as a means for preserving digital information. Inspired by the dynamic, biological regulation of (epi)genetic information, we herein present how binary data can undergo controlled changes when encoded in synthetic DNA strands. By exploiting differential kinetics of hydrolytic deamination reactions of cytosine and its naturally occurring derivatives, we demonstrate how multiple layers of information can be stored in a single DNA template. Moreover, we show that controlled redox reactions allow for interconversion of these DNA-encoded layers of information. Overall, such interlacing of multiple messages on synthetic DNA libraries showcases the potential of chemical reactions to manipulate digital information on (bio)polymers.C.M. is grateful for the financial support by the Swiss National Science Foundation (grant number P2EZP2_152216). G.R.M. was supported by funding from Trinity College, Cambridge, the Herchel Smith fund and the Wellcome Trust. P.M. was funded by the Wellcome Trust and is currently supported by an ERC Advanced grant. P.V.D was funded by the Wellcome Trust and a Marie Curie Fellow of the European Union (grant number FP7-PEOPLE-2013-IEF/624885). The S.B. lab is supported by a program grant and core funding from Cancer Research UK (C9681/A18618), an ERC Advanced grant (339778) and by a Senior Investigator Award of the Wellcome Trust (099232/Z/12/Z). We thank Eun-Ang Raiber and Dario Beraldi for stimulating discussions and proofreading the manuscript.This is the final version of the article. It first appeared from Wiley at http://dx.doi.org/10.1002/anie.201605531