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Regulation of DNA Replication Timing

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演者 Nick Rhind
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School (Professor)
会場 東京都医学総合研究所 2階講堂
日時 平成30年9月18日(火)16:00~
世話人 正井 久雄 (東京都医学総合研究所 所長)
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講演要旨

Eukaryotic genomes replicate in defined patterns, with some loci replicating early in S phase and others replicating later. Replication timing correlates with transcription, chromatin modification, sub-nuclear localization and genome evolution, suggesting an intimate association between replication timing and other important aspects of chromosome metabolism. However, the mechanism that regulates replication timing is currently unknown.

We present a comprehensive explanation for the regulation of replication timing in budding yeast. We propose that origin timing is regulated by the number of MCM complexes loaded at an origin, with origins that have more MCMs loaded having a higher probability of firing and thus an earlier average replication time and origins that have fewer MCMs loaded having a later average replication time. We have tested this assertion by measuring the level of MCM loading at each origin by MCM ChIP-seq. As predicted, we find a strong correlation between MCM ChIP-seq signal and replication timing. Moreover, we have directly measured the number of MCMs loaded at specific origins by affinity purification of single-origin plasmids and find that early-replicating plasmid origins load more MCMs that late replicating ones. Finally, by manipulating the number of MCMs loaded at chromosomal origins through cis-acting ARS mutations, we can affect the timing of origin firing.We propose that the loading of MCMs is regulated by the ORC occupancy at origins during G1, with origins that bind ORC for a greater fraction of G1 having more MCMs loaded. Consistent with this hypothesis, we see a strong correlation between MCM ChIP-seq signal and ORC ChIP-seq signal at origins. We are currently manipulating ORC occupancy at specific origins to test this model.

In addition to the ORC-regulated, MCM-loading-based regulation of origin timing, heterochromatin can affect origin timing. Our preliminary results suggest that this regulation occurs largely at the level of MCM activation. Heterochromatic origins load as many MCM complexes as non-heterochromatic loci, but the heterochromatic MCMs are much less likely to fire than MCMs loaded at euchromatic origins.

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