Spatiotemporal kinetics of CAF-1-dependent chromatin maturation ensures transcription fidelity during S-phase.

Proper maintenance of epigenetic information after replication is dependent on the rapid assembly and maturation of chromatin. Chromatin Assembly Complex 1 (CAF-1) is a conserved histone chaperone that deposits (H3-H4) tetramers as part of the replication-dependent chromatin assembly process. Loss of CAF-1 leads to a delay in chromatin maturation, albeit with minimal impact on steady-state chromatin structure.

Spatiotemporal kinetics of CAF-1-dependent chromatin maturation ensures transcription fidelity during S-phase.

Proper maintenance of epigenetic information after replication is dependent on the rapid assembly and maturation of chromatin. Chromatin Assembly Complex 1 (CAF-1) is a conserved histone chaperone that deposits (H3-H4) tetramers as part of the replication-dependent chromatin assembly process. Loss of CAF-1 leads to a delay in chromatin maturation, albeit with minimal impact on steady-state chromatin structure.

Disruption of origin chromatin structure by helicase activation in the absence of DNA replication.

Prior to initiation of DNA replication, the eukaryotic helicase, Mcm2-7, must be activated to unwind DNA at replication start sites in early S phase. To study helicase activation within origin chromatin, we constructed a conditional mutant of the polymerase α subunit Cdc17 (or Pol1) to prevent priming and block replication. Recovery of these cells at permissive conditions resulted in the generation of unreplicated gaps at origins, likely due to helicase activation prior to replication initiation.

Linking the dynamics of chromatin occupancy and transcription with predictive models.

Though the sequence of the genome within each eukaryotic cell is essentially fixed, it exists within a complex and changing chromatin state. This state is determined, in part, by the dynamic binding of proteins to the DNA. These proteins-including histones, transcription factors (TFs), and polymerases-interact with one another, the genome, and other molecules to allow the chromatin to adopt one of exceedingly many possible configurations.

Local nucleosome dynamics and eviction following a double-strand break are reversible by NHEJ-mediated repair in the absence of DNA replication.

We interrogated at nucleotide resolution the spatiotemporal order of chromatin changes that occur immediately following a site-specific double-strand break (DSB) upstream of the locus and its subsequent repair by nonhomologous end joining (NHEJ). We observed the immediate eviction of a nucleosome flanking the break and the repositioning of adjacent nucleosomes away from the break. These early chromatin events were independent of the end-processing Mre11-Rad50-Xrs2 (MRX) complex and preceded the MRX-dependent broad eviction of histones and DNA end-resectioning that extends up to ∼8 kb away from the break.

Nascent chromatin occupancy profiling reveals locus- and factor-specific chromatin maturation dynamics behind the DNA replication fork.

Proper regulation and maintenance of the epigenome is necessary to preserve genome function. However, in every cell division, the epigenetic state is disassembled and then reassembled in the wake of the DNA replication fork. Chromatin restoration on nascent DNA is a complex and regulated process that includes nucleosome assembly and remodeling, deposition of histone variants, and the re-establishment of transcription factor binding.

Mcm2-7 Is an Active Player in the DNA Replication Checkpoint Signaling Cascade via Proposed Modulation of Its DNA Gate.

The DNA replication checkpoint (DRC) monitors and responds to stalled replication forks to prevent genomic instability. How core replication factors integrate into this phosphorylation cascade is incompletely understood. Here, through analysis of a unique mcm allele targeting a specific ATPase active site (mcm2DENQ), we show that the Mcm2-7 replicative helicase has a novel DRC function as part of the signal transduction cascade.

Genome-wide chromatin footprinting reveals changes in replication origin architecture induced by pre-RC assembly.

Start sites of DNA replication are marked by the origin recognition complex (ORC), which coordinates Mcm2-7 helicase loading to form the prereplicative complex (pre-RC). Although pre-RC assembly is well characterized in vitro, the process is poorly understood within the local chromatin environment surrounding replication origins. To reveal how the chromatin architecture modulates origin selection and activation, we "footprinted" nucleosomes, transcription factors, and replication proteins at multiple points during the Saccharomyces cerevisiae cell cycle.

Dynamic loading and redistribution of the Mcm2-7 helicase complex through the cell cycle.

Eukaryotic replication origins are defined by the ORC-dependent loading of the Mcm2-7 helicase complex onto chromatin in G1. Paradoxically, there is a vast excess of Mcm2-7 relative to ORC assembled onto chromatin in G1. These excess Mcm2-7 complexes exhibit little co-localization with ORC or replication foci and can function as dormant origins.

Heterogeneous polymerase fidelity and mismatch repair bias genome variation and composition.

Mutational heterogeneity must be taken into account when reconstructing evolutionary histories, calibrating molecular clocks, and predicting links between genes and disease. Selective pressures and various DNA transactions have been invoked to explain the heterogeneous distribution of genetic variation between species, within populations, and in tissue-specific tumors. To examine relationships between such heterogeneity and variations in leading- and lagging-strand replication fidelity and mismatch repair, we accumulated 40,000 spontaneous mutations in eight diploid yeast strains in the absence of selective pressure.

Genome-wide localization of replication factors.

Chromatin Immunoprecipitation (ChIP) is a powerful tool for the identification and characterization of protein-DNA interactions in vivo. ChIP has been utilized to study diverse nuclear processes such as transcription regulation, chromatin modification, DNA recombination and DNA replication at specific loci and, more recently, across the entire genome. Advances in genomic approaches, and whole genome sequencing in particular, have made it possible and affordable to comprehensively identify specific protein binding sites throughout the genomes of higher eukaryotes.

Identification of functional elements and regulatory circuits by Drosophila modENCODE.

To gain insight into how genomic information is translated into cellular and developmental programs, the Drosophila model organism Encyclopedia of DNA Elements (modENCODE) project is comprehensively mapping transcripts, histone modifications, chromosomal proteins, transcription factors, replication proteins and intermediates, and nucleosome properties across a developmental time course and in multiple cell lines. We have generated more than 700 data sets and discovered protein-coding, noncoding, RNA regulatory, replication, and chromatin elements, more than tripling the annotated portion of the Drosophila genome. Correlated activity patterns of these elements reveal a functional regulatory network, which predicts putative new functions for genes, reveals stage- and tissue-specific regulators, and enables gene-expression prediction.

Chromatin signatures of the Drosophila replication program.

DNA replication initiates from thousands of start sites throughout the Drosophila genome and must be coordinated with other ongoing nuclear processes such as transcription to ensure genetic and epigenetic inheritance. Considerable progress has been made toward understanding how chromatin modifications regulate the transcription program; in contrast, we know relatively little about the role of the chromatin landscape in defining how start sites of DNA replication are selected and regulated. Here, we describe the Drosophila replication program in the context of the chromatin and transcription landscape for multiple cell lines using data generated by the modENCODE consortium.

Drosophila ORC localizes to open chromatin and marks sites of cohesin complex loading.

The origin recognition complex (ORC) is an essential DNA replication initiation factor conserved in all eukaryotes. In Saccharomyces cerevisiae, ORC binds to specific DNA elements; however, in higher eukaryotes, ORC exhibits little sequence specificity in vitro or in vivo. We investigated the genome-wide distribution of ORC in Drosophila and found that ORC localizes to specific chromosomal locations in the absence of any discernible simple motif.