Monitoring the compaction of single DNA molecules in Xenopus egg extract in real time
- Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720
- Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, Institute for Quantitative Biosciences (QB3), University of California, Berkeley, CA 94720
- Institute for Quantitative Biosciences (QB3), University of California, Berkeley, CA 94720
- Chromosome Dynamics Laboratory, RIKEN, Wako 351-0198, Japan
- Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, Institute for Quantitative Biosciences (QB3), University of California, Berkeley, CA 94720, Department of Physics, University of California, Berkeley, CA 94720, Department of Chemistry, University of California, Berkeley, CA 94720, HHMI, University of California, Berkeley, CA 94720
DNA compaction is required for the condensation and resolution of chromosomes during mitosis, but the relative contribution of individual chromatin factors to this process is poorly understood. We developed a physiological, cell-free system using high-speed Xenopus egg extracts and optical tweezers to investigate real-time mitotic chromatin fiber formation and force-induced disassembly on single DNA molecules. Compared to interphase extract, which compacted DNA by ~60%, metaphase extract reduced DNA length by over 90%, reflecting differences in whole-chromosome morphology under these two conditions. Depletion of the core histone chaperone ASF1, which inhibits nucleosome assembly, decreased the final degree of metaphase fiber compaction by 29%, while depletion of linker histone H1 had a greater effect, reducing total compaction by 40%. Compared to controls, both depletions reduced the rate of compaction, led to more short periods of decompaction, and increased the speed of force-induced fiber disassembly. In contrast, depletion of condensin from metaphase extract strongly inhibited fiber assembly, resulting in transient compaction events that were rapidly reversed under high force. Altogether, these findings support a speculative model in which condensin plays the predominant role in mitotic DNA compaction, while core and linker histones act to reduce slippage during loop extrusion and modulate the degree of DNA compaction.
- Research Organization:
- Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
- Sponsoring Organization:
- Japan Society for the Promotion of Science (JSPS); National Institutes of Health (NIH); USDOE Office of Science (SC), Basic Energy Sciences (BES)
- Grant/Contract Number:
- AC02-05CH11231
- OSTI ID:
- 1961479
- Alternate ID(s):
- OSTI ID: 2422587
- Journal Information:
- Proceedings of the National Academy of Sciences of the United States of America, Journal Name: Proceedings of the National Academy of Sciences of the United States of America Journal Issue: 12 Vol. 120; ISSN 0027-8424
- Publisher:
- Proceedings of the National Academy of SciencesCopyright Statement
- Country of Publication:
- United States
- Language:
- English
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