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Abstract
Replication-coupled nucleosome assembly is a critical step in packaging newly synthesized DNA into chromatin. Previous studies have defined the importance of the histone chaperones CAF-1 and ASF1A, the replicative clamp PCNA, and the clamp loader RFC for the assembly of nucleosomes during DNA replication. Despite significant progress in the field, replication-coupled nucleosome assembly is not well understood. One of the complications in elucidating the mechanisms of replication-coupled nucleosome assembly is the lack of a defined system that faithfully recapitulates this important biological process in vitro. We describe here a defined system that assembles nucleosomal arrays in a manner dependent on the presence of CAF-1, ASF1A-H3-H4, H2A-H2B, PCNA, RFC, NAP1L1, ATP, and strand breaks. The loss of CAF-1 p48 subunit causes a strong defect in packaging DNA into nucleosomes by this system. We also show that the defined system forms nucleosomes on nascent DNA synthesized by the replicative polymerase δ. Thus, the developed system reproduces several key features of replication-coupled nucleosome assembly.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
We are grateful to Dr Hitoshi Kurumizaka for the NAP1L1 overexpression plasmid, Dr Erich Nigg for ASF1A plasmid, Dr Danny Reinberg for SPT16 and SSRP1 plasmids, and Farid F Kadyrov for critical reading of the manuscript. This work was supported by start-up funds from the Southern Illinois University School of Medicine and NIH grant R01GM095758 (to FAK). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.