Regulation of Replication Fork Progression Through Histone Supply and Demand

Abstract

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.

DNA Looping Mediates Nucleosome Transfer

“…Consistent with this requirement, overexpression of new histones or perturbation of chaperone function both result in replication fork stalling 9 . This implies that when daughter DNA is saturated with new histones, or new histones are not positioned properly, the parental nucleosome at the fork cannot be efficiently transferred and therefore becomes a substantial barrier for replication.…”

“…Current views suggest that histone chaperones and nucleosome assembly factors act in a coordinated manner at the replication fork to shuttle parental histones and deposit nascent histones to newly replicated DNA 2,9 . However, this theory is only loosely described and neglects the role of DNA itself, the fundamental component of nucleosome organization and dynamics.…”

Therapeutic targeting of constitutive PARP activation compromises stem cell phenotype and survival of glioblastoma-initiating cells

“…We next evaluated the homeostatic levels of single-strand DNA (ssDNA) in matched GICs and non-GICs as assessed by BrDU incorporation under non-denaturing conditions and detected enhanced ssDNA in GIC populations (Supplementary Figure 3b). 34,40,41 We also used the alkaline comet assay to measure DNA strand breaks. GICs had significantly longer tails and higher comet tail DNA content as compared with the non-GICs, indicating the extent of fragmented DNA at baseline was greater in the GICs ( Supplementary Figure 3c-e).…”

“…Detection of gH2AX (1 : 500; Abcam, Cambridge, UK) was performed as described previously. 34,40 GICs and non-GICs were grown on GelTrex (Gibco, Invitrogen, Carlsbad, CA, USA)-coated cover slips and treated with DMSO or 10 mM olaparib and left unirradiated or irradiated with 3 Gy and fixed 1, 6, or 24 h after irradiation. Cells were then immunostained for gH2AX.…”

Histones, histone chaperones and nucleosome assembly

“…Structural studies have revealed that Asf1 binds H3-H4 dimers through an H3 interface that is involved in the formation of (H3-H4) 2 tetramers (English et al, 2006). In human cells, it is proposed that Asf1 can disrupt parental nucleosomes (Groth et al, 2007). Furthermore, Asf1 is required for acetylation of histone H3 lysine 56 (H3K56Ac), a mark of newly-synthesized histones that has been found to be important for DNA replication and DNA repair (Recht et al, 2006;Chen et al, 2008;Li et al, 2008).…”