Orc4 spatiotemporally stabilizes centromeric chromatin

Abstract

words) 1Spatiotemporal regulation in DNA replication maintains kinetochore stability. The epigenetically 2 regulated centromeres (CENs) in the budding yeast Candida albicans have unique DNA sequences, 3 replicate early and are clustered throughout the cell cycle. In this study, the genome-wide occupancy 4 of replication initiation protein Orc4 reveals its abundance at all CENs in C. albicans. Orc4 associates 5 with four different DNA motifs, one of which coincides with tRNA genes. Hi-C combined with 6 genome-wide replication timing analyses identify enriched interactions among early or late replicating 7Orc4-bound regions. A simulated polymer model of chromosomes reveals that early replicating and 8 strongly enriched Orc4-bound sites localize towards the kinetochores. Orc4 is constitutively localized 9to CENs, and both Orc4 and Mcm2 stabilize CENPA. CENPA chaperone Scm3 localizes at the 10 kinetochore during anaphase, coinciding with the loading time of CENPA. We propose that this 11 spatiotemporal nuclear localization of Orc4, with Mcm2 and Scm3, recruits CENPA and stabilizes 12 centromeric chromatin.

Cellular Dynamics and Genomic Identity of Centromeres in Cereal Blast Fungus

“…Rather, non-DNA sequence determinants mark centromeres in an epigenetic manner in many organisms. Some epigenetic determinants of centromere identity in fungi include early replicating regions of the genome (71–73), proximity to DNA replication origins (15), DNA replication initiator proteins (74), homologous recombination-repair proteins (15, 75), and proteins that facilitate kinetochore clustering by tethering kinetochores to SPBs (31, 65). Factors that favor local folding and looping of chromatin may also add to the process of centromere specification (4, 14, 76).…”

Cellular Dynamics and Genomic Identity of Centromeres in the Cereal Blast Fungus

“…Rather, non-DNA sequence determinants mark centromeres in an epigenetic manner in many organisms. Some epigenetic determinants of centromere identity in fungi include early replicating regions of the genome [61-63], proximity to DNA replication origins [64], DNA replication initiator proteins [65], homologous recombination-repair proteins [64, 66] and proteins that facilitate kinetochore clustering by tethering kinetochores to SPBs [22, 54]. Factors that favor local folding and looping of chromatin may also add to the process of centromere specification [4, 52, 67].…”

A phylogenetically-restricted essential cell cycle progression factor in the human pathogen Candida albicans

“…Overexpression of genes from the tetracyline inducible promoter (PTET) was achieved by the addition of anhydrotetracycline (Atc, 3 µg/ml) or doxycycline (Dox, 50 µg/ml) in YPDU medium at 30°C (47) in the dark as Atc and Dox are light-sensitive. The CSA6 PSD strains were grown at 30°C either in permissive (YPDU) or nonpermissive (YPDU + 5mM methionine (M) + 5mM cysteine (C)) conditions of the MET3 promoter (59,61). E. coli strains were cultured at 30°C or 37°C in Luria-Bertani (LB) medium or 2YT supplemented with ampicillin (50 µg/ml or 100 µg/ml), chloramphenicol (34 µg/ml), kanamycin (50 µg/ml) and tetracycline (10 µg/ml).…”

“…In a recent study from our laboratory, Mcm7 has been identified as a subunit of the kinetochore interactome in a basidiomycete yeast Cryptococcus neoformans(89). Another subunit of the Mcm2-7 complex, Mcm2, is involved in regulating the stability of centromeric chromatin in C. albicans(61). Considering the growing evidence of the role of Mcm2-7 subunits beyond their canonical, well-established roles in DNA replication, the serendipitous identification of Mcm7 as a regulator of genome stability in our screen is striking.…”