Biology subjects

Griffin, P.

Publications and source records attributed to Griffin, P..

At least 19 recordsLinked to original sources

bioRxiv2026

Development of low cost, robust and reproducible biofilm static and pharmacodynamic assays

SynopsisO_ST_ABSBackgroundC_ST_ABSThe complexity of diagnosing and treating biofilm-associated infections necessitates a comprehensive strategy to mitigate the rising rates of antimicrobial resistance (AMR). Microtiter plate methods are used globally for determination of biofilm eradication concentrations (MBEC) but few have been adapted to observe pharmacodynamic observations. Here, we describe a method which allows for both static and pharmacodynamic assays of biofilm evaluation. MethodsA total of 150 clinical isolates from Southmead Hospital were assessed, representing five bacterial species (N=30 per bacterial species): Pseudomonas aeruginosa, Escherichia coli, Streptococcus pneumoniae, Staphylococcus aureus and Klebsiella pneumoniae. MBECs were determined using a developed method using 96 well plates and glass beads. MBECs of seven different antibiotics were compared to those determined using the established Calgary biofilm device (CBD). Dynamic pharmacodynamic evaluations to produce Biofilm Time Kill curve (BTKC) based on published planktonic time kill curve (TKC) data and ISO recommendations were carried out using the glass bead model for K. pneumoniae and ciprofloxacin, S. aureus and levofloxacin and S. pneumoniae and vancomycin. Quantification of biofilm biomass was assessed at 0, 2, 4, 8 and 24 hours and compared to planktonic culture survival under comparable challenge conditions. ResultsComparing MBEC results for all bacterial strains and antibiotic challenges showed no statistical difference between the glass bead and CBD methods (P <0.05). Biofilm BTKC AUBKC were inferior to planktonic equivalents but demonstrated specific pharmacodynamic patterns of biofilm reduction efficacy. MBEC correlated with biofilm BTKC penetration in line with clinical observations for S. aureus vs vancomycin and S. pneumoniae vs levofloxacin. ConclusionsThe glass bead biofilm models provide robust, reproducible alternatives to the traditional methods of determining MBEC and bridge the gap with biofilm pharmacodynamic evaluations. These methods also provide a low-cost option to current methods as only standard laboratory equipment is required, allowing for the generation of comprehensive data sets. This ensures greater translatability to complex in vitro models and clinical scenarios.

microbiology↗

bioRxiv2026

Implementing considered elements of standardisation for Time Kill Curve experiments across multiple sites: A European collaboration perspective

SynopsisO_ST_ABSBackgroundC_ST_ABSThe main advantages of Time Kill Curves (TKCs) in antimicrobial drug development are the ability to track bacterial kill and regrowth over time and with varying drug concentrations. Whilst there are guideline documents in place, such as M26-A in CLSI, there remains scope for individual laboratory differences in practice. Here we evaluated several factors which potentially influenced data generated in TKCs. MethodsFirstly, E. coli ATCC 25922 was used to determine optimum sampling volume, culture vessel volume, CFU enumeration variance factors and static versus agitated cultures in a single laboratory. Secondly, a ring test comprising of TKCs was performed by six laboratories focusing on: standardised inoculum, static culture and two culture vessel sizes 10 mL and 200 {micro}L. Data analysis was performed to determine consistency within centres and between them. ResultsConsistently accurate inocula could be achieved by use of: larger sampling volumes between 100 {micro}L > 20 mL; larger culture vessels volumes (10 mL > 100 {micro}L) and higher inocula (10 8 > 1.5x10 5 CFU). Culture agitation during the TKC experiment resulted in reduced killing compared to static cultures. Reproducibility of TKCs was best between centres when they were performed in 10 mL culture vessels. There was more variability per site when performing TKC in 96 well trays. ConclusionsTechnical factors such as preparation of inocula, agitation, vessel size and enumeration of cultures are important variables in performing TKCs that need to be standardised in drug development programmes involving multiple laboratory centres.

microbiology↗

bioRxiv2026

Is there a need to implement standardisation into in vitro antimicrobial evaluation systems? A European collaboration perspective

SynopsisO_ST_ABSBackgroundC_ST_ABSTime kill curve (TKC) assessments are an essential step in the study of an antimicrobials pharmacodynamic characteristics. Surprisingly TKCs have not be formally standardised, therefore there remain concerns that different testing centres/methodologies may produce different results. Six centres participating in Gram-negative-Antibiotics NOW (GNA-NOW) consortium measured a series of TKCs with meropenem against E. coli to establish: Same-day (SD) vs different-day (DD) replication per centre (intra-site), and centre to centre (inter-site) correlations. MethodsMeropenem was tested against three strains of E. coli (ATCC 25922; ESBL producer C1.55; OXA-48 producer C1.62). An inoculum of 1.5x106 CFU was specified with meropenem concentrations of x0, x1 to x16 MIC; and sampling assessment of bacterial density was determined at 0-24h. Experiments were performed in triplicate, aerobically at 37{degrees}C. Centre-specific methodology was collected. Meropenem, media, bacterial strains, were shipped from one central laboratory to participating laboratories. ANOVA and Friedman tests were used to assess SD, DD and between centre replications. ResultsAssessment of the methodologies between centres revealed many differences, including bacterial inoculum, meropenem preparation, volume of TKC vessel, vessel materials, agitation vs static cultures and sampling volumes. Intra-centre SD and DD analysis for all strains were generally associated with P>0.05 suggesting consistency. Inter-centre SD and DD comparisons resulted in P<0.05, indicating variable total bacterial load measurement between centres. ConclusionsTKC methodologies varied between different centres, and while intra-centre comparison of SD and DD were generally consistent, inter-centre comparisons were not. Standardisation of TKC methodologies is required.

microbiology↗

bioRxiv2026

AI-derived Protein Structures Validation: AlphaFold2 Models in the Twilight Zone

In any field, unquestioningly accepting artificial intelligence (AI) results should be considered bad practise. Here, we devised a comparative modelling-based strategy for validating protein structures that exploits the well-known observation that protein folds are far more conserved than protein sequences. We identify proteins with a similar fold to the AlphaFold-generated query protein and determine their structural alignment to the query. The hypothesis is that if the sequence alignment coincides with the structural alignment, then the structure is validated. The strategy is implemented on a helix-by-helix and strand-by-strand basis using a multi-template pairwise local profile alignment method that works well into the twilight zone. The method is illustrated by application to the transmembrane transporter PEPT1, for which the structure is known, and the S-deacylases ABHD13 and ABHD16A, for which only AI-generated models exist. ABHD16A is particularly challenging because a sequence alignment search with BLASTp does not reveal any structural homologues and therefore requires work with extremely remote homologues; however, both models are validated through this strategy and are stable during classical molecular dynamics simulations. The ability of the strategy to identify errors is assessed with reference to misaligned ABHD13 models and misfolded decoy proteins.

bioinformatics↗

bioRxiv2025

Bacteriophage pharmacodynamics studied in an in vitro pharmacokinetic model of infection

SynopsisO_ST_ABSBackgroundC_ST_ABSBacteriophage therapy offers an alternative way to counter the menace of increasing antimicrobial resistance. Despite its use in clinical practice for many decades the basic tools to study the translational pharmacodynamics of phages are not available and it is recognised that lack of understanding of phage pharmacokinetic/dynamics (PK/PD) is a severe limitation in individual patient use and clinical trial design. MethodsTraditional in vitro PK/PD evaluation tools were used to assess the antibacterial effect of single exposures of a bacteriophage cocktail against 4 strains of E. coli with potentially different patterns of response to phage. Initially, time-kill curves (TKC) were performed over 48hr and subsequently a dilutional in vitro model (IVM) was used to assess the antibacterial effects over 72hr. ResultsIn TKC, the four E. coli strains showed different patterns of kill and regrowth when exposed to phage with two strains showing a sustained drop in bacterial viable count and two showing initial kill and regrowth. Using the IVM similar bacterial pharmacodynamic patterns were observed, and phage titre increased inversely but consistently with E. coli kill. ConclusionsAn In vitro dilutional model can be used to study the antibacterial effect of a phage cocktail on E.coli showing strain-to-strain variation in bacterial killing and bacteriophage titre. Such models can be used to provide more nuanced information on phage pharmacokinetics/dynamics and translationally useful information for dosing in humans.

microbiology↗

bioRxiv2024

Development of antibacterial drug plus bacteriophage combination assays

SynopsisO_ST_ABSBackgroundC_ST_ABSThe methods to evaluate the interactions between Phages and antibacterials are unclear. As the laboratory methodologies used to assess conventional antibacterials are well established, we asseassed their efficacy in evaluating phage plus antibacterial. Methods100 multidrug resistant E. coli strains were used with three previously isolated and characterised E. coli phages of known efficacy. These phages UP17, JK08, 113 were assessed both individually and in a 1:1:1cocktail. In a Phage Microbial Inhibitory Concentration (PmIC) assay, a range of phage concentrations from 101 -108 were inoculated with 5x105/well bacteria in microtitre plates. The first lysed, clear well was taken as the PmIC. Amikacin(AMI) and meropenem(MERO) MICs were determined by microbroth dilution methods(ISO 2776-1:2019) and in combination AMI and MERO MICs were measured with a fixed Phage concentration of 105/well. MICs were performed in triplicate. Time-Kill curves(TKC) were conducted at fosfomycin concentrations of 133, 50 and 5mg/L with and without phage. ResultsThe PmIC50/90 for UP17 were >108/>108; JK08 107/>108; 113 107/>108 and the 1:1:1 cocktail 106/>108. AMI MIC50/90 were 0.5/>16 and MERO 0.12/>16mg/L. The addition of UP17 to AMI increased AMI MICs >2 fold in 78 strains. Equivalent increases in AMI MIC were seen with 39 strains with JK08, 54 strains with 113 and 45 strains with the cocktails. In contrast, meropenem MICs in the presence of phage were reduced >2 fold in 24 strains with UP17. Equivalent decreases in MERO MIC were seen with 34 strains with JK08, 26 strains with 113 and 29 strains with the cocktails. In TKCs addition of phage suppressed regrowth. ConclusionMicrobroth methodologies based on ISO 2776-1:2019 and TKCs allow the interaction between Phages and antibacterials to be studied. Optimisation may produce laboratory-based methods with translational value.

microbiology↗

bioRxiv2022

Hidden GPCR structural transitions addressed by multiple walker supervised molecular dynamics (mwSuMD)

The structural basis for the pharmacology of G protein-coupled receptors (GPCRs), the most abundant membrane proteins and the target of about 35% of approved drugs, is still a matter of intense study. What makes GPCRs challenging to study is the inherent flexibility and the metastable nature of interaction with extra-and intracellular partners that drive their effects. Here, we present a molecular dynamics (MD) adaptive sampling algorithm, namely multiple walker supervised molecular dynamics (mwSuMD), to address complex structural transitions involving GPCRs without energy input. We first report the binding and unbinding of the vasopressin peptide from its receptor V2. Successively, we present the complete transition of the glucagon-like peptide-1 receptor (GLP-1R) from inactive to active, agonist and Gs-bound state, and the GDP release from Gs. To our knowledge, this is the first time the whole sequence of events leading from an inactive GPCR to the GDP release is simulated without any energy bias. We demonstrate that mwSuMD can address complex binding processes intrinsically linked to protein dynamics out of reach of classic MD.

bioinformatics↗

bioRxiv2021

Ultrastructural insight into SARS-CoV-2 attachment, entry and budding in human airway epithelium

Ultrastructural studies of SARS-CoV-2 infected cells are crucial to better understand the mechanisms of viral entry and budding within host cells. Many studies are limited by the lack of access to appropriate cellular models. As the airway epithelium is the primary site of infection it is essential to study SARS-CoV-2 infection of these cells. Here, we examined human airway epithelium, grown as highly differentiated air-liquid interface cultures and infected with three different isolates of SARS-CoV-2 including the B.1.1.7 variant (Variant of Concern 202012/01) by transmission electron microscopy and tomography. For all isolates, the virus infected ciliated but not goblet epithelial cells. Two key SARS-CoV-2 entry molecules, ACE2 and TMPRSS2, were found to be localised to the plasma membrane including microvilli but excluded from cilia. Consistent with these observations, extracellular virions were frequently seen associated with microvilli and the apical plasma membrane but rarely with ciliary membranes. Profiles indicative of viral fusion at the apical plasma membrane demonstrate that the plasma membrane is one site of entry where direct fusion releasing the nucleoprotein-encapsidated genome occurs. Intact intracellular virions were found within ciliated cells in compartments with a single membrane bearing S glycoprotein. Profiles strongly suggesting viral budding from the membrane was observed in these compartments and this may explain how virions gain their S glycoprotein containing envelope.

microbiology↗

bioRxiv2021

Structural insights into the role of Dicer-related helicase 3 in RNAi in Caenorhabditis elegans.

DRH-3 belongs to the family of duplex RNA-dependent ATPases (DRAs), which include Dicer and RIG-I-like receptors (RLRs). DRH-3 is critically involved in germline development and RNAi-facilitated chromosome segregation via the 22G-siRNA pathway in C. elegans. The molecular understanding of DRH-3 and its function in endogenous RNAi pathways remains elusive. In this study, we solved the crystal structures of the DRH-3 N-terminal domain (NTD) and the C-terminal domains (CTDs) in complex with 5-triphosphorylated RNAs. The NTD of DRH-3 adopts a distinct fold of tandem Caspase Activation and Recruitment Domains (CARDs) structurally similar to the CARDs of RIG-I and MDA5, suggesting a signaling function in the endogenous RNAi biogenesis. The CTD preferentially recognizes 5-triphosphorylated double-stranded RNAs bearing the typical features of secondary siRNA transcripts. The full-length DRH-3 displays unique structural dynamics upon binding to RNA duplexes that differ from RIG-I or MDA5. These unique molecular features of DRH-3 help explain its function in RNAi in worms and the evolutionary divergence of the Dicer-like helicases.

biochemistry↗

bioRxiv2020

PPARG in osteocytes is essential for sclerostin expression, bone mass, marrow adiposity and TZD-induced bone loss

PPARG role in regulation of osteocyte function is largely unknown. We report that PPARG is essential for sclerostin production, a recently approved target to treat osteoporosis. There is an excellent correlation in osteocytes between Sost/sclerostin and PPARG at the transcript and protein levels, and increased bone mass in mice with osteocyte-specific deletion of PPARG ({gamma}OTKO) correlated with increased WNT signaling and bone forming activity of endosteal osteoblasts and decreased marrow fat. The 8 kb sequence upstream of Sost gene transcription start site possesses multiple PPARG binding elements (PPREs) with at least two of them binding PPARG with dynamics reflecting its activation and the levels of Sost transcript and sclerostin protein expression. Older {gamma}OTKO female mice are largely protected from TZD-induced bone loss providing proof of concept that PPARG in osteocytes can be pharmacologically targeted. Our study opens the possibility to consider repurposing PPARG as a target for treatment of osteoporosis.

physiology↗

bioRxiv2020

Discovery of a Novel, Selective and Short-Acting Skeletal Myosin II Inhibitor

Myosin IIs, actin-based motors that utilize the chemical energy of ATP to generate force, have potential as therapeutic targets. Their heavy chains differentiate the family into muscle (skeletal [SkMII], cardiac, smooth) and nonmuscle myosin IIs. Despite therapeutic potential for muscle disorders, no SkMII-specific inhibitor has been reported and characterized. Here we present the discovery, synthesis and characterization of "skeletostatins", novel derivatives of the pan-myosin II inhibitor blebbistatin, with selectivity within the myosin IIs for SkMII. In addition, the skeletostatins bear improved potency, solubility and photostability, without cytotoxicity. Based on its optimal in vitro profile, Skeletostatin 1s in vivo tolerability, efficacy and pharmacokinetics were determined. Skeletostatin 1 was well-tolerated in mice, impaired motor performance, and had an excellent muscle to plasma ratio. Skeletostatins are useful probes for basic research and a strong starting point for drug development.

pharmacology and toxicology↗

bioRxiv2020

Dual-Mechanism Estrogen Receptor Inhibitors Reveal an Ensemble of Binding Modes for Robust Breast Cancer Antagonism

Efforts to improve estrogen receptor-a (ER)-targeted therapies in breast cancer have relied upon a single mechanism, with ligands having a single side chain on the ligand core that extends outward to determine antagonism of breast cancer growth. Here, we describe inhibitors with two ER-targeting moieties, one of which uses an alternate structural mechanism to generate full antagonism, freeing the side chain to independently determine other critical properties of the ligands. By combining two molecular targeting approaches into a single ER ligand, we have generated antiestrogens that function through new mechanisms and structural paradigms to achieve antagonism. These dual-mechanism ER inhibitors (DMERIs) cause alternate, non-canonical structural perturbations of the receptor ligand-binding domain (LBD) to drive antagonism of proliferation in ER-positive breast cancer cells and in allele-specific resistance models. Solution structural and coregulator peptide binding analyses with DMERIs highlight marked differences from current standard-of-care, single-mechanism antiestrogens. These findings uncover an enhanced flexibility of the ER LBD through which it can access non-consensus conformational modes in response to DMERI binding, broadly and effectively suppressing ER activity. Significance StatementTo address the unmet clinical need for effectively suppressing estrogen receptor (ER) activity with both de novo resistance and in advanced ER-positive breast cancers that are resistant to standard-of-care antiestrogens, we have developed dual-mechanism ER inhibitors (DMERIs) that employ two distinct ER targeting moieties. These DMERI elicited non-canonical structural perturbations of the receptor ligand-binding domain and stabilized multiple antagonist sub-states within the dimer to generate highly efficacious antagonism of proliferation in ER-positive breast cancer cells and in allele-specific resistance models. This work reveals new conformational modes by which the activity of ER can be effectively suppressed to block breast cancer proliferation.

cancer biology↗

bioRxiv2020

REV-ERBalpha mediates complement expression and circadian regulation of microglial synaptic phagocytosis

The circadian clock has been shown to regulate various aspects of brain health including microglial and astrocyte activation. Here we report that deletion of the master clock protein BMAL1 induces robust increases in the expression of complement genes such as C3, C4b and C1q in the hippocampus. Loss of downstream REV-ERB-mediated transcriptional repression led to increases in C4b in neurons and astrocytes as well as C3 protein in microglia and astrocytes. REV-ERB deletion induced complement C3/C4b gene expression and increased microglial phagocytosis of synapses in the CA3 region of the hippocampus. Finally, we observed diurnal variation in the degree of microglial synaptic phagocytosis in wild type mice which was abrogated by REV-ERB deletion. This work uncovers the BMAL1-REV-ERB axis as a regulator of complement expression and synaptic phagocytosis in the brain, thereby illuminating a novel mechanism of synaptic regulation by the circadian clock.

neuroscience↗

bioRxiv2020

A Simple and Robust Cell Based Assay for the Discovery of Novel Cytokinesis Inhibitors

Cytokinesis is the last step of mitotic cell division that separates the cytoplasm of dividing cells. Small molecule inhibitors targeting either the elements of the regulatory pathways controlling cytokinesis, or the terminal effectors have been of interest as potential drug candidates for the treatment of various diseases. Here we have developed a cell-based assay for the discovery of novel cytokinesis inhibitors. The assay is performed in a 96-well plate format in 48 hours. Living cells, nuclei and nuclei of dead cells are revealed by a single staining step with three fluorescent dyes, followed by live cell imaging using an automated fluorescence microscope. The primary signal is the nuclei-to-cell ratio (NCR). In the presence of cytokinesis inhibitors, this ratio is expected to increase over time, as the ratio of multinucleated cells increases in the population. Cytotoxicity is quantified as the ratio of dead nuclei to total nuclei. A screening window coefficient (Z) of 0.65 indicates that the assay is suitable for screening purposes, as the positive and negative controls are well-separated. EC50 values for four compounds can be reliably determined in a single 96-well plate by using only six different compound concentrations. An excellent test-retest reliability (R2=0.998) was found for EC50 values covering a [~]1500-fold range of potencies. The robustness, simplicity and flexibility of the assay is demonstrated here by using modulators of actin dynamics with different mechanisms of action (jasplakinolide, cytochalasin D and swinholide A) and derivatives of the nonmuscle myosin II inhibitor blebbistatin.

cell biology↗

bioRxiv2019

Overcoming high nanopore basecaller error rates for DNA storage via basecaller-decoder integration and convolutional codes

As magnetization and semiconductor based storage technologies approach their limits, bio-molecules, such as DNA, have been identified as promising media for future storage systems, due to their high storage density (petabytes/gram) and long-term durability (thousands of years). Furthermore, nanopore DNA sequencing enables high-throughput sequencing using devices as small as a USB thumb drive and thus is ideally suited for DNA storage applications. Due to the high insertion/deletion error rates associated with basecalled nanopore reads, current approaches rely heavily on consensus among multiple reads and thus incur very high reading costs. We propose a novel approach which overcomes the high error rates in basecalled sequences by integrating a Viterbi error correction decoder with the basecaller, enabling the decoder to exploit the soft information available in the deep learning based basecaller pipeline. Using convolutional codes for error correction, we experimentally observed 3x lower reading costs than the state-of-the-art techniques at comparable writing costs. The code, data and Supplementary Material is available at https://github.com/shubhamchandak94/nanopore_dna_storage.

bioengineering↗

bioRxiv2019

Improved read/write cost tradeoff in DNA-based data storage using LDPC codes

With the amount of data being stored increasing rapidly, there is significant interest in exploring alternative storage technologies. In this context, DNA-based storage systems can offer significantly higher storage densities (petabytes/gram) and durability (thousands of years) than current technologies. Specifically, DNA has been found to be stable over extended periods of time which has been demonstrated in the analysis of organisms long since extinct. Recent advances in DNA sequencing and synthesis pipelines have made DNA-based storage a promising candidate for the storage technology of the future.\n\nRecently, there have been multiple efforts in this direction, focusing on aspects such as error correction for synthesis/sequencing errors and erasure correction for handling missing sequences. The typical approach is to use separate codes for handling errors and erasures, but there is limited understanding of the efficiency of this framework. Furthermore, the existing techniques use short block-length codes and heavily rely on read consensus, both of which are known to be suboptimal in coding theory.\n\nIn this work, we study the tradeoff between the writing and reading costs involved in DNA-based storage and propose a practical scheme to achieve an improved tradeoff between these quantities. Our scheme breaks with the traditional separation framework and instead uses a single large block-length LDPC code for both erasure and error correction. We also introduce novel techniques to handle insertion and deletion errors introduced by the synthesis process. For a range of writing costs, the proposed scheme achieves 30-40% lower reading costs than state-of-the-art techniques on experimental data obtained using array synthesis and Illumina sequencing.\n\nThe code, data, and Supplementary Material is available at https://github.com/shubhamchandak94/LDPC_DNA_storage.

bioengineering↗

bioRxiv2019

Quantitative Structural Assessment of Graded Receptor Agonism

Ligand-receptor interactions, which are ubiquitous in physiology, are described by theoretical models of receptor pharmacology. Structural evidence for graded-efficacy receptor conformations predicted by receptor theory has been limited, but is critical to fully validate theoretical models. We applied quantitative structure-function approaches to characterize the effects of structurally similar and structurally diverse agonists on the conformational ensemble of nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR{gamma}). For all ligands, agonist efficacy is correlated to a shift in the conformational ensemble equilibrium from a ground state towards an active state, which is detected by NMR spectroscopy but not observed in crystal structures. For the structurally similar ligands, ligand potency is also correlated to efficacy and conformation, indicating ligand residence times among related analogs can influence receptor conformation and function. Our results derived from quantitative graded activity-conformation correlations provide new experimental evidence and a platform with which to extend and test theoretical models of receptor pharmacology to more accurately describe and predict ligand-dependent receptor activity.

biophysics↗

bioRxiv2019

Chromosomal rearrangements at hypomethylated Satellite 2 sequences are associated with impaired replication efficiency and increased fork stalling

Cancer cells, aging cells, and cells from patients with the developmental disorder Immunodeficiency, Centromeric instability, and Facial anomalies (ICF) syndrome frequently display a striking loss of DNA methylation (hypomethylation) that is accompanied by increased DNA damage and chromosomal rearrangements. Despite the robust link, the mechanism by which hypomethylation leads to genomic instability is poorly understood. We report that the human pericentromeric repeat sequence Satellite 2 (SAT2) poses challenges to the DNA replication machinery when hypomethylated. Loss of methylation at SAT2 is associated with increased frequencies of chromosomal abnormalities and DNA damage. Hypomethylation of SAT2 is associated with elevated levels of replication stress signaling, and chromosomal abnormalities involving SAT2 are enhanced by low levels of aphidicolin-induced replication stress. To investigate the basis for these chromosomal abnormalities, we developed a single-molecule approach employing DNA combing to examine the progress of replication forks through SAT2 at the resolution of a single DNA molecule. Our analysis of replicating SAT2 molecules provides in vivo evidence that hypomethylation of SAT2 strongly decreases the efficiency of replicating these sequences suggesting that hypomethylation results in the formation of barriers to the replication machinery. Consistent with increased frequency of fork stalling at these sequences, we find increased levels of single-stranded DNA (ssDNA) binding protein RPA2 as well as asymmetric progression of sister replication forks within hypomethylated SAT2 sequences. Together these findings indicate that impaired replication triggers the formation of chromosomal aberrations observed at hypomethylated SAT2 sequences and also suggests a mechanistic basis for how the loss of DNA methylation may contribute to genomic instability in diverse pathological conditions.

molecular biology↗