Replisome loading reduces chromatin motion independent of DNA synthesis.

Chromatin has been shown to undergo diffusional motion, which is affected during gene transcription by RNA polymerase activity. However, the relationship between chromatin mobility and other genomic processes remains unclear. Hence, we set out to label the DNA directly in a sequence unbiased manner and followed labeled chromatin dynamics in interphase human cells expressing GFP-tagged proliferating cell nuclear antigen (PCNA), a cell cycle marker and core component of the DNA replication machinery.

Deep probabilistic tracking of particles in fluorescence microscopy images.

Tracking of particles in temporal fluorescence microscopy image sequences is of fundamental importance to quantify dynamic processes of intracellular structures as well as virus structures. We introduce a probabilistic deep learning approach for fluorescent particle tracking, which is based on a recurrent neural network that mimics classical Bayesian filtering. Compared to previous deep learning methods for particle tracking, our approach takes into account uncertainty, both aleatoric and epistemic uncertainty.

Developmental differences in genome replication program and origin activation.

To ensure error-free duplication of all (epi)genetic information once per cell cycle, DNA replication follows a cell type and developmental stage specific spatio-temporal program. Here, we analyze the spatio-temporal DNA replication progression in (un)differentiated mouse embryonic stem (mES) cells. Whereas telomeres replicate throughout S-phase, we observe mid S-phase replication of (peri)centromeric heterochromatin in mES cells, which switches to late S-phase replication upon differentiation.

Processive DNA synthesis is associated with localized decompaction of constitutive heterochromatin at the sites of DNA replication and repair.

Constitutive heterochromatin is considered as a functionally inert genome compartment, important for its architecture and stability. How such stable structure is maintained is not well understood. Here, we apply four different visualization schemes to label it and investigate its dynamics during DNA replication and repair.

Preferable location of chromosomes 1, 29, and X in bovine spermatozoa.

Chromosome positioning in sperm nucleus may have a functional significance by influencing the sequence of post-fertilization events. In this study we present data on preferential locations of chromosomes 1, 29 and X in spermatozoa. Here we demonstrate that the position of X chromosome in the sperm nucleus is more restricted as compared to the position of chromosome 1, which is about of the same size.

4D Visualization of replication foci in mammalian cells corresponding to individual replicons.

Since the pioneering proposal of the replicon model of DNA replication 50 years ago, the predicted replicons have not been identified and quantified at the cellular level. Here, we combine conventional and super-resolution microscopy of replication sites in live and fixed cells with computational image analysis. We complement these data with genome size measurements, comprehensive analysis of S-phase dynamics and quantification of replication fork speed and replicon size in human and mouse cells.

3D replicon distributions arise from stochastic initiation and domino-like DNA replication progression.

DNA replication dynamics in cells from higher eukaryotes follows very complex but highly efficient mechanisms. However, the principles behind initiation of potential replication origins and emergence of typical patterns of nuclear replication sites remain unclear. Here, we propose a comprehensive model of DNA replication in human cells that is based on stochastic, proximity-induced replication initiation.

DNA double-strand break repair is impaired in presenescent Syrian hamster fibroblasts.

Background: Studies of DNA damage response are critical for the comprehensive understanding of age-related changes in cells, tissues and organisms. Syrian hamster cells halt proliferation and become presenescent after several passages in standard conditions of cultivation due to what is known as "culture stress". Using proliferating young and non-dividing presenescent cells in primary cultures of Syrian hamster fibroblasts, we defined their response to the action of radiomimetic drug bleomycin (BL) that induces DNA double-strand breaks (DSBs).

High-resolution analysis of Mammalian DNA replication units.

Genomic DNA of a eukaryotic cell is replicated once during the S-phase of the cell cycle to precisely maintain the complete genetic information. In the course of S-phase, semiconservative DNA synthesis is sequentially initiated and performed at thousands of discrete patches of the DNA helix termed replicons. At any given moment of S-phase, multiple replicons are active in parallel in different parts of the genome.

Organization of DNA replication.

The discovery of the DNA double helix structure half a century ago immediately suggested a mechanism for its duplication by semi-conservative copying of the nucleotide sequence into two DNA daughter strands. Shortly after, a second fundamental step toward the elucidation of the mechanism of DNA replication was taken with the isolation of the first enzyme able to polymerize DNA from a template. In the subsequent years, the basic mechanism of DNA replication and its enzymatic machinery components were elucidated, mostly through genetic approaches and in vitro biochemistry.

Cell segmentation in time-lapse fluorescence microscopy with temporally varying sub-cellular fusion protein patterns.

Fluorescently tagged proteins such as GFP-PCNA produce rich dynamically varying textural patterns of foci distributed in the nucleus. This enables the behavioral study of sub-cellular structures during different phases of the cell cycle. The varying punctuate patterns of fluorescence, drastic changes in SNR, shape and position during mitosis and abundance of touching cells, however, require more sophisticated algorithms for reliable automatic cell segmentation and lineage analysis.

Measurement of replication structures at the nanometer scale using super-resolution light microscopy.

DNA replication, similar to other cellular processes, occurs within dynamic macromolecular structures. Any comprehensive understanding ultimately requires quantitative data to establish and test models of genome duplication. We used two different super-resolution light microscopy techniques to directly measure and compare the size and numbers of replication foci in mammalian cells.

Segmentation and classification of cell cycle phases in fluorescence imaging.

Current chemical biology methods for studying spatiotemporal correlation between biochemical networks and cell cycle phase progression in live-cells typically use fluorescence-based imaging of fusion proteins. Stable cell lines expressing fluorescently tagged protein GFP-PCNA produce rich, dynamically varying sub-cellular foci patterns characterizing the cell cycle phases, including the progress during the S-phase. Variable fluorescence patterns, drastic changes in SNR, shape and position changes and abundance of touching cells require sophisticated algorithms for reliable automatic segmentation and cell cycle classification.

Inhibition of transcription at radiation-induced nuclear foci of phosphorylated histone H2AX in mammalian cells.

Double-strand DNA breaks (DSBs) induced by ionizing radiation can be visualized in human cells using antibodies against Ser-139 phosphorylated histone H2AX (gamma-H2AX). Large gamma-H2AX foci are seen in the nucleus fixed 1 hour after irradiation and their number corresponds to the number of DSBs, allowing analysis of these genome lesions after low doses. We estimated whether transcription is affected in chromatin domains containing gamma-H2AX by following in vivo incorporation of 5-bromouridine triphosphate (BrUTP) loaded by cell scratching (run-on assay).

[High resolution analysis of replication foci by conventional fluorescent microscopy. I. A study of complexity and DNA content of the foci].

Newly replicated DNA segments (RDS) have been shown to form discrete foci in the mammalian nucleus. Comparison of the number of such foci in formaldehyde-fixed cell nucleus with estimated number of simultaneously active replication forks (RF) suggests that each replication focus contains a cluster of about 10 to 20 closely associated RF. That implied the cluster of synchronously activated replicons as the primary unit of mammalian DNA replication.

Conformation of replicated segments of chromosome fibres in human S-phase nucleus.

Recent statistical analysis of the folding of G0/G1 chromosomes using fluorescence in situ hybridization (FISH) allowed development of a random walk/giant loop model of chromosome structure. According to this model there are two levels of organization of G0/G1 chromosome fibres. On the first level, the fibres are arranged in giant loops several Mbp in size, and within each loop the fibres are randomly folded.

[Organization of DNA replication domains in S-phase nuclei of human cells].

Each chromosome of eukaryotic cells contains multiple units of DNA replication that are activated during S-phase of cell cycle according to a definite program. It is considered at present that the main independent units of replication in mammalian cells represent groups of 20-25 adjacent synchronously activated small (with the average size 100 kbp) replicons. After labelling of nascent DNA with nonradioactive DNA precursors and immunofluorescent staining of incorporated label, discrete replication domains (RDs) are detected in S-phase nuclei.

Staurosporine-sensitive protein phosphorylation is required for postreplication DNA repair in human cells.

DNA repair is an important factor of stability of pro- and eukaryotic genomes which plays a central role in mutagenesis and carcinogenesis. Genetic control of nucleotide excision repair (NER) in mammalian cells is well studied, but little is known about molecular mechanisms of postreplication repair (PRR) which allows bypass of base lesions in template strands after DNA replication. In Saccharomyces cerevisiae PRR is controlled by the RAD61RAD18 pathway which involves POL30 gene encoding proliferating cell nuclear antigen (PCNA), and in human cells PCNA is known to be closely associated with the newly replicated chromatin where PRR probably takes place.