The histone chaperone ASF1 localizes to active DNA replication forks to mediate efficient DNA replication

Abstract

The packaging of the eukaryotic genome into chromatin is likely to regulate all processes that occur on the DNA template. The assembly and disassembly of chromatin structures from histone proteins and DNA are mediated by histone chaperones, including the histone H3/H4 chaperone anti-silencing function 1 (ASF1). To address the function of ASF1 in metazoan cells, we used RNA interference-mediated knockdown of Drosophila melanogaster ASF1 (dASF1). Cells lacking dASF1 accumulate in S phase of the cell cycle as determined by flow cytometry analysis of DNA content and quantitation of the proportion of cells with replication foci. In agreement, bromodeoxyuridine (BrdU) pulse-chase analysis demonstrates that the absence of ASF1 leads to delayed progression through S-phase. Furthermore, the absence of ASF1 leads to a reduced ability to incorporate the nucleoside analog BrdU, indicating that ASF1 is required for efficient DNA replication. We have also found that dASF1 colocalizes with DNA replication foci throughout S phase by immunofluorescence analysis and that these dASF1 foci are disrupted upon inhibition of DNA replication by treatment of cells with hydroxyurea. As such, these results demonstrate that dASF1 is present at active, but not stalled, replication forks. We propose that dASF1 has a direct role in modifying chromatin structure during DNA replication and that this function of dASF1 is important for the processivity of the replication machinery.

ANTI-SILENCING FUNCTION1 Proteins Are Involved in Ultraviolet-Induced DNA Damage Repair and Are Cell Cycle Regulated by E2F Transcription Factors in Arabidopsis

“…ASF1 is also required to efficiently complete DNA replication in the presence of DNA-damaging agents or compromised replication machinery (Franco et al, 2005). In Drosophila melanogaster, ASF1 was shown to assemble chromatin onto newly replicated DNA in vitro in synergy with CAF1 (Tyler et al, 1999) and to colocalize with active replication forks (Schulz and Tyler, 2006). Even though information on Arabidopsis ASF1 is limited, the results obtained so far suggest that this histone chaperone is involved in cell cycle regulation.…”

Remodeling of chromatin structure in senescent cells and its potential impact on tumor suppression and aging

“…Yeast Asf1p is required for heterochromatin-mediated silencing of telomeres and mating loci and has histone deposition activity in vitro (67,116,121,127). Yeast Asf1p is also required for histone eviction and subsequent replacement at transcribed genes (1,66,111), and also plays a role in DNA replication-coupled chromatin assembly (44,51,86,110,127). Yeast Hir1p and Hir2p share several biological and biochemical properties with Asf1p.…”

The Histone Chaperone Anti-silencing Function 1 Stimulates the Acetylation of Newly Synthesized Histone H3 in S-phase

“…It is known that when this process is coupled to DNA replication in vitro, it is mediated by the histone H3/H4 chaperone chromatin assembly factor 1 (CAF-1) 2 (5) together with another H3/H4 chaperone termed anti-silencing function 1 (Asf1) (6). CAF-1 and Asf1 have also been shown to localize to the sites of DNA replication (7,8), supporting a role in assembling chromatin following DNA replication in vivo. The fact that CAF-1 and Asf1 co-purify with the replicationspecific histone variant H3.1 from HeLa cells is further evidence that Asf1 and CAF-1 function together to assemble newly replicated DNA into chromatin (9).…”