An essential role for Orc6 in DNA replication through maintenance of pre-replicative complexes

Abstract

The heterohexameric origin recognition complex (ORC) acts as a scaffold for the G 1 phase assembly of pre-replicative complexes (pre-RC). Only the Orc1-5 subunits appear to be required for origin binding in budding yeast, yet Orc6 is an essential protein for cell proliferation. Imaging of Orc6-YFP in live cells revealed a punctate pattern consistent with the organization of replication origins into subnuclear foci. Orc6 was not detected at the site of division between mother and daughter cells, in contrast to observations for metazoans, and is not required for mitosis or cytokinesis. An essential role for Orc6 in DNA replication was identified by depleting it at specific cell cycle stages. Interestingly, Orc6 was required for entry into S phase after pre-RC formation, in contrast to previous models suggesting ORC is dispensable at this point in the cell cycle. When Orc6 was depleted in late G 1 , Mcm2 and Mcm10 were displaced from chromatin, cells failed to progress through S phase, and DNA combing analysis following bromodeoxyuridine incorporation revealed that the efficiency of replication origin firing was severely compromised.

Defining replication origin efficiency using DNA fiber assays

“…These authors were also able to reveal subtle initiation defects in cells defective for the CDK inhibitor Sic1, which were hardly detectable with classical biochemical assays (Lengronne and Schwob 2002). Stronger initiation defects were also measured in various initiation mutants using this approach (Devault et al 2002;Semple et al 2006;Shimada et al 2002). .…”

“…However, it is important to note that initiation rates can be overestimated when IODs are measured in cells presenting severe initiation defects. For instance, depletion of the Orc6 subunit in GAL-ORC6 cells increases IODs by 70% (Semple et al 2006), but this analysis does not take into account DNA fibers containing a single initiation event (Fig. 3d).…”

“…Medians are indicated in kilobases; d schematic representation of the distribution of active origins (filled circles) in GAL-ORC6 cells upon Orc6 depletion. IODs are indicated in kilobases; e comparison of IODs (filled boxes) and the ratio of origins to total DNA length (empty boxes) measured in wild type and GAL-ORC6 cells (Semple et al 2006). Boxes 25-75 percentile range.…”

Structural analysis of human Orc6 protein reveals a homology with transcription factor TFIIB

“…Drosophila Orc6 has a DNA binding activity on its own, with a specificity that can account for the poly(dA) preference of ORC binding (19). Recently, an evidence appeared for the role of Orc6 in pre-RC formation in budding yeast, where Orc6 directly interacts with Cdt1 during repeated Mcm2-7 loading (21,22). Orc6 in budding yeast is significantly larger than in metazoan species and it remains to be investigated whether Orc6 is important for pre-RC formation in higher eukaryotes.…”

Helicase Loading at Chromosomal Origins of Replication

“…Consistent with this observation, in vitro studies show that the helicase loading proteins are released from the origin upon Mcm2 -7 loading (Randell et al 2006;Tsakraklides and Bell 2010). In contrast to these findings, in vivo inactivation of S. cerevisiae ORC or Cdc6 in late G 1 (after Mcm2 -7 loading) results in loss of Mcm2 -7originassociation(Aparicioetal.1997;Semple et al 2006;Chen et al 2007). This suggests either that these proteins are required to maintain Mcm2 -7 association with the origin DNA or that there is an activity that removes loaded Mcm2 -7 from the DNA in G 1 cells and the helicase loading proteins are required to restore Mcm2 -7 to the origin.…”