Regulation of Replication Fork Progression Through Histone Supply and Demand

Laboratory of Nuclear Dynamics and Genome Plasticity, UMR218 CNRS/Institut Curie, 26 rue d'Ulm, 75248 Paris cedex 05, France.
Science (Impact Factor: 33.61). 01/2008; 318(5858):1928-31. DOI: 10.1126/science.1148992
Source: PubMed


DNA replication in eukaryotes requires nucleosome disruption ahead of the replication fork and reassembly behind. An unresolved
issue concerns how histone dynamics are coordinated with fork progression to maintain chromosomal stability. Here, we characterize
a complex in which the human histone chaperone Asf1 and MCM2–7, the putative replicative helicase, are connected through a
histone H3-H4 bridge. Depletion of Asf1 by RNA interference impedes DNA unwinding at replication sites, and similar defects
arise from overproduction of new histone H3-H4 that compromises Asf1 function. These data link Asf1 chaperone function, histone
supply, and replicative unwinding of DNA in chromatin. We propose that Asf1, as a histone acceptor and donor, handles parental
and new histones at the replication fork via an Asf1–(H3-H4)–MCM2–7 intermediate and thus provides a means to fine-tune replication
fork progression and histone supply and demand.

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    • "ASF1 binds a heterodimeric histone H3-H4 complex through its conserved N-terminal domain (aa 1–155) and impedes the formation of the (H3-H4) 2 tetramer both inside the nucleus and in the cytoplasm (English et al., 2005). Humans have two ASF1 paralogs, ASF1a and ASF1b, both of which can be co-purified with the MCM 2-7 replicative helicase and histones H3 and H4 from HeLa cell nuclear extract, while only the MCM2 subunit can be pulled down by ASF1a or ASF1b together with H3 and H4 when using cytosolic extract of the same cells (Groth et al., 2007). In vitro studies showed that an N-terminal of human MCM2 (aa 63–154) binds histone H3 directly through a conserved motif (Foltman et al., 2013; Ishimi et al., 1998). "

    Full-text · Article · Jul 2015 · Protein & Cell
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    • "We considered several possibilities to explain the apparent simultaneous DNA occupancy of Mcm2–7 and the flanking nucleosome (see the Results), but our data were most consistent with a nucleosome being pulled down together with the immunoprecipitated Mcm2–7 complex. Prior experiments have reported that certain Mcm2–7 subunits directly interact with histone H3 (Ishimi et al. 1996), and the Mcm2–7 complex mediates histone contacts with FACT and Asf1 at replication forks (Groth et al. 2007;Foltman et al. 2013). We propose that the origin-flanking nucleosomes both positionally confine and engage with Mcm2–7 in vivo to facilitate subsequent unwinding of the origin DNA in S phase. "
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    ABSTRACT: 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. Our nucleotide-resolution protein occupancy profiles resolved a precise ORC-dependent footprint at 269 origins in G2. A separate class of inefficient origins exhibited protein occupancy only in G1, suggesting that stable ORC chromatin association in G2 is a determinant of origin efficiency. G1 nucleosome remodeling concomitant with pre-RC assembly expanded the origin nucleosome-free region and enhanced activation efficiency. Finally, the local chromatin environment restricts the loading of the Mcm2-7 double hexamer either upstream of or downstream from the ARS consensus sequence (ACS). © 2015 Belsky et al.; Published by Cold Spring Harbor Laboratory Press.
    Preview · Article · Jan 2015 · Genes & Development
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    • "This distribution pattern is similar to endogenous RPA as shown by immunofluorescence (Fig. 1B). Sub-nuclear structures with higher local concentrations were independent of the cell cycle phase and were previously described to co-localize with promyelocytic leukemia (PML) body markers [33] [34]. "
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    ABSTRACT: The ability of replication protein A (RPA) to bind single-stranded DNA (ssDNA) underlines its crucial roles during DNA replication and repair. A combination of immunofluorescence and live cell imaging of GFP-tagged RPA70 revealed that RPA, in contrast to other replication factors, does not cluster into replication foci, which is explained by its short residence time at ssDNA. In addition to replication, RPA also plays a crucial role in both the pre- and post-incision steps of nucleotide excision repair (NER). Pre-incision factors like XPC and TFIIH accumulate rapidly at locally induced UV-damage and remain visible up to 4 h. However, RPA did not reach its maximum accumulation level until 3 h after DNA damage infliction and a chromatin-bound pool remained detectable up to 8 h, probably reflecting its role during the post-incision step of NER. During the pre-incision steps of NER, RPA could only be visualized at DNA lesions in incision deficient XP-F cells, however without a substantial increase in residence time at DNA damage. Together our data show that RPA is an intrinsically highly dynamic ssDNA-binding complex during both replication and distinct steps of NER.
    Full-text · Article · Dec 2014 · DNA Repair
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