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The RCAF complex mediates chromatin assembly during DNA replication and repair

Abstract

Chromatin assembly is a fundamental biological process that is essential for the replication and maintenance of the eukaryotic genome1,2,3,4. In dividing cells, newly synthesized DNA is rapidly assembled into chromatin by the deposition of a tetramer of the histone proteins H3 and H4, followed by the deposition of two dimers of histones H2A and H2B to complete the nucleosome—the fundamental repeating unit of chromatin5. Here we describe the identification, purification, cloning, and characterization of replication-coupling assembly factor (RCAF), a novel protein complex that facilitates the assembly of nucleosomes onto newly replicated DNA in vitro. RCAF comprises the Drosophila homologue of anti-silencing function 1 protein ASF16 and histones H3 and H4. The specific acetylation pattern of H3 and H4 in RCAF is identical to that of newly synthesized histones. Genetic analyses in Saccharomyces cerevisiae demonstrate that ASF1 is essential for normal cell cycle progression, and suggest that RCAF mediates chromatin assembly after DNA replication and the repair of double-strand DNA damage in vivo.

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Figure 1: Purification of RCAF activity.
Figure 2: RCAF cannot be replaced by histones and acts synergistically with CAF-1 in the assembly of chromatin on to newly replicated DNA.
Figure 3: Defects in growth and transcriptional silencing after the loss of both RCAF and CAF-1 activity.
Figure 4: Effects of disruptions to ASF1 and CAC1 on sensitivity to mutagens.

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Acknowledgements

We thank P. Willy, D. Fyodorov, X. Huang, J. Butler, M. Levenstein, N. Dhillon, D. Donze, S. Ghidelli, L. Krushel and E. Stone for critical reading of the manuscript; L. Pillus for helpful suggestions and discussions; T. Laverty and G. Rubin for determination of the cytological location of dASF1; K. Collins for generating recombinant dASF1 protein; M. Levenstein for H3 antibodies and purified histones H3–H4; D. Allis for H4 antibodies; and R. Sternglanz, P. Kaufman, D. Shore, D. Gottschling and J. Rine for providing various strains and plasmids used in this study. This work was supported by grants from the NIH to J.T.K., R.K. and R.T.K.; J.K.T. is a Leukemia Society of America Special Fellow.

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Correspondence to Jessica K. Tyler.

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Tyler, J., Adams, C., Chen, SR. et al. The RCAF complex mediates chromatin assembly during DNA replication and repair. Nature 402, 555–560 (1999). https://doi.org/10.1038/990147

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