Cyan fluorescent proteins (CFPs), such as Cerulean, are widely used as donor fluorophores in Förster resonance energy transfer (FRET) experiments. Nonetheless, the most widely used variants suffer from drawbacks that include low quantum yields and unstable flurorescence. To improve the fluorescence properties of Cerulean, we used the X-ray structure to rationally target specific amino acids for optimization by site-directed mutagenesis. Optimization of residues in strands 7 and 8 of the β-barrel improved the quantum yield of Cerulean from 0.48 to 0.60. Further optimization by incorporating the wild-type T65S mutation in the chromophore improved the quantum yield to 0.87. This variant, mCerulean3, is 20% brighter and shows greatly reduced fluorescence photoswitching behavior compared to the recently described mTurquoise fluorescent protein in vitro and in living cells. The fluorescence lifetime of mCerulean3 also fits to a single exponential time constant, making mCerulean3 a suitable choice for fluorescence lifetime microscopy experiments. Furthermore, inclusion of mCerulean3 in a fusion protein with mVenus produced FRET ratios with less variance than mTurquoise-containing fusions in living cells. Thus, mCerulean3 is a bright, photostable cyan fluorescent protein which possesses several characteristics that are highly desirable for FRET experiments.

Cycling cells duplicate their DNA content during S phase, following a defined program called replication timing (RT). Early and late replicating regions differ in terms of mutation rates, transcriptional activity, chromatin marks and sub-nuclear position. Moreover, RT is regulated during development and is altered in diseases such as leukemia. Here, we describe E/L repli-seq, an extension of our repli-chip protocol. E/L repli-seq is a rapid, robust and relatively inexpensive protocol to analyze RT by next-generation sequencing (NGS), allowing genome-wide assessment of how cellular processes are linked to RT. Briefly, cells are pulse labeled with BrdU and early and late S phase fractions are sorted by flow cytometry. Labeled nascent DNA is immunoprecipitated from both fractions and sequenced. Data processing leads to a single bedGraph file containing the ratio of nascent DNA from early versus late S phase fractions. The results are comparable to repli-chip, with the additional benefits of genome-wide sequence information and an increased dynamic range. We also provide computational pipelines for downstream analyses, for parsing phased genomes using single nucleotide polymorphisms (SNP) to analyze RT allelic asynchrony, and for direct comparison to repli-chip data. This protocol can be performed in up to three days prior to sequencing, and requires basic cellular and molecular biology skills and a basic understanding of Unix and R.

Components of the minichromosome maintenance complex (Mcm2-7) remain indefinitely bound to chromatin during G1 phase and replication arrest.

All eukaryotic cells replicate segments of their genomes in a defined temporal sequence. In multicellular organisms, at least half of the genome is subject to changes in this temporal sequence during development. We now know that this temporal sequence and its developmentally regulated changes are conserved across distantly related species, suggesting that it either represents or reflects something biologically important. However, both the mechanism and the significance of this program remain unknown. We recently demonstrated a remarkably strong genome-wide correlation between replication timing and chromatin interaction maps, stronger than any other chromosomal property analyzed to date, indicating that sequences localized close to one another replicate at similar times. This provides molecular confirmation of long-standing cytogenetic evidence for spatial compartmentalization of early- and late-replicating DNA and supports our earlier model that replication timing is reestablished in each G(1) phase, coincident with the anchorage of chromosomal segments at specific locations within the nucleus (timing decision point [TDP]). Here, we review the evidence linking the replication program to the three-dimensional architecture of chromatin in the nucleus and discuss what such a link might mean for the mechanism and significance of a developmentally regulated replication program.