Fork pausing allows centromere DNA loop formation and kinetochore assembly

Abstract

De novo kinetochore assembly, but not template-directed assembly, is dependent on COMA, the kinetochore complex engaged in cohesin recruitment. The slowing of replication fork progression by treatment with phleomycin (PHL), hydroxyurea, or deletion of the replication fork protection protein Csm3 can activate de novo kinetochore assembly in COMA mutants. Centromere DNA looping at the site of de novo kinetochore assembly can be detected shortly after exposure to PHL. Using simulations to explore the thermodynamics of DNA loops, we propose that loop formation is disfavored during bidirectional replication fork migration. One function of replication fork stalling upon encounters with DNA damage or other blockades may be to allow time for thermal fluctuations of the DNA chain to explore numerous configurations. Biasing thermodynamics provides a mechanism to facilitate macromolecular assembly, DNA repair, and other nucleic acid transactions at the replication fork. These loop configurations are essential for sister centromere separation and kinetochore assembly in the absence of the COMA complex.

Replication fork pausing at protein barriers on chromosomes

“…The modest pausing of replication forks at centromeres observed in wild‐type cells was increased when Rrm3 was deleted , and double deletion of Pif1 and Rrm3 enhanced the pausing . Fork pausing or slowing by the treatment of yeast cells with phleomycin or hydroxyurea restored centromere DNA loop formation and kinetochore assembly in mutants of the centromere‐associated protein genes . Accordingly, a specific system appears to exist that slows down the forks at the centromere to establish the site‐specific function, and Rrm3 and Pif1 play a role in controlling the system with respect to modulating fork speed.…”

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].…”

Common Features of the Pericentromere and Nucleolus

“…While CTCF has not been identified in lower eukaryotic species such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Caenorhabditis elegans [94], transformation studies suggest that CTCF has a similar insulating function in yeast [95]. Furthermore, members of the Ctf19/COMA complex, which regulates pericentromeric cohesin enrichment [51,96,97], play a vital role pericentromere loop formation [98], perhaps similar to mechanism by which CTCF mediates cohesin at the base of loops [99]. This suggests that the Ctf19/COMA complex could function as the yeast equivalent of CTCF.…”

“…The lack of breakage that is normally induced in a dicentric strain is consistent with a lack of de novo kinetochore assembly in these mutants. Furthermore, de novo kinetochore assembly in Chl4/Iml3 mutants is rescued by pausing replication using hydroxyurea [98]. By slowing the replication process, this may allow additional time for these mutants to resume proper kinetochore assembly.…”