A pinboard by
Aftab Amin

Post Doctoral Fellow, Hong Kong University of Science and Technology


An essentail cell cycle-dependent and semi-conservative ORC ‘dimerization cycle was uncovered

Eukaryotic DNA replication licensing, a prerequisite for genome duplication, plays a critical role to ensure that all chromosomal DNA is replicated exactly once per cell cycle. It involves the recruitment of replication-initiation proteins by origin recognition complex (ORC) to form pre-replicative complexes (pre-RCs) at replication origins. ORC binds to and marks replication origins throughout the cell cycle. However, the regulation of ORC in replication licensing has not been fully elucidated. Here we report that ORC self-interacts and dimerizes (forming a double heterohexamer) before pre-RC formation in budding yeast. Upon S phase entry, each double hexamer ORC separates into two single hexamers to bind each pair of nascent origins until late M phase. The non-chromatin-bound ORC then associates with the chromatin-bound ORC to reform double hexamers at the M-to-G1 transition. Preventing ORC dimerization by depleting non-chromatin bound ORC proteins in M phase, or by Orc6p point mutations that disrupt ORC self-interaction but can maintain ORC inter-subunit interactions, abolishes pre-RC formation, DNA replication and cell proliferation and viability. Similarly, human ORC also self interacts. Moreover, hORC co-localizes on stretched human chromatin fiber bundles in a cell cycle dependent manner: occurs in G1 phase but not in G2/M phase. Our findings uncovered an essential, cell cycle-dependent and semi-conservative ORC ‘dimerization cycle’ that plays three fundamental roles in the regulation of DNA replication: providing a symmetric platform to load the symmetric pre-RCs, marking and protecting the nascent sister replication origins until the next licensing, and providing the first guard against origin re-licensing within the same cell cycle.


A Role of hIPI3 in DNA Replication Licensing in Human Cells.

Abstract: The yeast Ipi3p is required for DNA replication and cell viability in Sacharomyces cerevisiae. It is an essential component of the Rix1 complex (Rix1p/Ipi2p-Ipi1p-Ipi3p) that is required for the processing of 35S pre-rRNA in pre-60S ribosomal particles and for the initiation of DNA replication. The human IPI3 homolog is WDR18 (WD repeat domain 18), which shares significant homology with yIpi3p. Here we report that knockdown of hIPI3 resulted in substantial defects in the chromatin association of the MCM complex, DNA replication, cell cycle progression and cell proliferation. Importantly, hIPI3 silencing did not result in a reduction of the protein level of hCDC6, hMCM7, or the ectopically expressed GFP protein, indicating that protein synthesis was not defective in the same time frame of the DNA replication and cell cycle defects. Furthermore, the mRNA and protein levels of hIPI3 fluctuate in the cell cycle, with the highest levels from M phase to early G1 phase, similar to other pre-replicative (pre-RC) proteins. Moreover, hIPI3 interacts with other replication-initiation proteins, co-localizes with hMCM7 in the nucleus, and is important for the nuclear localization of hMCM7. We also found that hIPI3 preferentially binds to the origins of DNA replication including those at the c-Myc, Lamin-B2 and β-Globin loci. These results indicate that hIPI3 is involved in human DNA replication licensing independent of its role in ribosome biogenesis.

Pub.: 09 Apr '16, Pinned: 28 Jul '17