DNA synthesis, we normalized MNase–BrdU-IP–
seq signals using sonication–BrdU-IP–seq signals
to derive a ReIN score, which is the quotient of
nascent nucleosome coverage based on the MNase–
BrdU-IP–seq signal over the nascent DNA coverage
represented by the sonication–BrdU-IP–seq signal.
To reduce the potential overestimation of ReIN
scores for bases with low sequencing coverage,
raw genome counts for sonication–BrdU-IP–seq
quantitation were simulated by smoothing based
on an exponential model (Fig. 4E, right). The
ReIN scores surrounding ACSs were reduced in
mutant cells, and the degree of reduction in rfa1-
A88P mutant cells was comparable to that of
rtt109D mutant cells (Fig. 4F). Thus, nucleosome
assembly on nascent chromatin is compromised
by disruption of the RPA–H3-H4 interaction.
In this study, we suggest that RPA, an essen-
tial replisome component, provides a binding
platform for histone H3-H4 and multiple histone
H3-H4 chaperones, including CAF-1, Rtt106, and
FACT. Our results support a model whereby RPA
binds ssDNA and promotes the deposition of
H3-H4 onto adjacent dsDNA, in part through
its ability to bind H3-H4. We observed that de-
oxyribonuclease I (DNase I) and ethidium bromide
treatment of cell lysates resulted in a stronger
RPA–H3-H4 interaction, implying that RPA may
bind parental histones released from chromatin
(22). Mcm2, a subunit of replicative helicase
MCM, contains a motif that binds H3-H4 and
is not required for DNA replication (23). MCM
(24), and the interactions between MCM and
histone chaperones Asf1 and FACT have been
proposed to facilitate nucleosome disassembly
of parental H3-H4 following DNA replication
(22, 24). RPA binds ssDNA immediately after
the unwinding of dsDNA by MCM, prior to DNA
synthesis (5). Given our finding that RPA func-
tions in nucleosome assembly on nascent chro-
matin, we suggest that RPA provides a common
binding platform for the coordination of histone
deposition by multiple histone chaperones dur-
ing RC nucleosome assembly.
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We thank Z. Zhang, R. Burgess, K.-M. Chan, and H. Wu for critical
reading of the manuscript. We thank M. Wold, O. Aparcio, R. Xu,
G. Li, S. Brill, M. Grunstein, G. Camilloni, and J. Tyler for reagents
and suggestions for experimental procedures. We thank the
Biodynamic Optical Imaging Center (BIOPIC) sequencing facility
at Peking University for assistance with sequencing. This work
was supported by National Natural Science Foundation of China
(NSFC) grants (31322017 and 31370767). K.C. is supported, in
part, by an NIH grant (HL100397). The high-throughput sequencing
data sets have been deposited at the National Center for
Biotechnology Information (NCBI) Gene Expression Omnibus
(GEO) database (accession identifier GSE83648).
Materials and Methods
Figs. S1 to S15
Tables S1 to S3
16 July 2016; accepted 21 December 2016
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