Histone chaperones that escort histones during their overall lifetime from 
synthesis to sites of usage can participate in various tasks. Their requirement 
culminates in the dynamic processes of nucleosome assembly and disassembly. In 
this context, it is important to define the exact role of the histone chaperone 
Asf1. In mammals, Asf1 interacts with two other chaperones, CAF-1 and HIRA, 
which are critical in DNA synthesis-coupled and synthesis-uncoupled nucleosome 
assembly pathways, respectively. A key issue is whether Asf1 is able or not to 
deposit histones onto DNA by itself in both pathways. Here, to delineate the 
precise role of Asf1 in chromatin assembly, we used Xenopus egg extracts as a 
powerful system to assay de novo chromatin assembly pathways in vitro. Following 
characterization of both Xenopus Asf1 and p60 (CAF-1), we used immunodepletion 
strategies targeting Asf1, HIRA, or CAF-1. Strikingly, the depletion of Asf1 led 
to the simultaneous depletion of HIRA and consequently impaired the DNA 
synthesis-independent nucleosome assembly pathway. The rescue of nucleosome 
assembly capacity in such extracts was effective when adding HIRA along with 
H3/H4 histones, yet addition of Asf1 along with H3/H4 histones did not work. 
Moreover, nucleosome assembly coupled to DNA repair was not affected in these 
Asf1/HIRA-depleted extracts, a pathway impaired by CAF-1 depletion. Thus, these 
data show that Asf1 is not directly involved in de novo histone deposition 
during DNA synthesis-independent and synthesis-dependent pathways in egg 
extracts. Based on our results, it becomes important to consider the 
implications for Asf1 function during early development in Xenopus.