The Hsk1(Cdc7) Replication Kinase Regulates Origin Efficiency

Abstract

Origins of DNA replication are generally inefficient, with most firing in fewer than half of cell cycles. However, neither the mechanism nor the importance of the regulation of origin efficiency is clear. In fission yeast, origin firing is stochastic, leading us to hypothesize that origin inefficiency and stochasticity are the result of a diffusible, rate-limiting activator. We show that the Hsk1-Dfp1 replication kinase (the fission yeast Cdc7-Dbf4 homologue) plays such a role. Increasing or decreasing Hsk1-Dfp1 levels correspondingly increases or decreases origin efficiency. Furthermore, tethering Hsk1-Dfp1 near an origin increases the efficiency of that origin, suggesting that the effective local concentration of Hsk1-Dfp1 regulates origin firing. Using photobleaching, we show that Hsk1-Dfp1 is freely diffusible in the nucleus. These results support a model in which the accessibility of replication origins to Hsk1-Dfp1 regulates origin efficiency and provides a potential mechanistic link between chromatin structure and replication timing. By manipulating Hsk1-Dfp1 levels, we show that increasing or decreasing origin firing rates leads to an increase in genomic instability, demonstrating the biological importance of appropriate origin efficiency.

Controlling DNA replication origins in response to DNA damage – inhibit globally, activate locally

“…Thus, although there is an excess of licensed origins, firing factors are limiting for replication. Consistent with this, overexpression of firing factors increases the efficiency of origin firing and can induce late origins to fire early (Krasinska et al, 2008;Patel et al, 2008;Wu and Nurse, 2009;Mantiero et al 2011;). Why firing factors have different affinities for different origins is currently unknown and has been discussed elsewhere (Douglas and Diffley, 2012).…”

“…In budding yeast, Sld3 and Cdc45 associate with early-firing origins early in G1 phase, whereas they do not associate with late-firing origins until approximately the time these origins fire in S phase (Aparicio et al, 1997;Gambus et al, 2006;Kamimura et al, 2001). Recent experiments suggest that the amount of firing factors present determines both the efficiency of origin firing and its timing (Krasinska et al, 2008;Mantiero et al, 2011;Patel et al, 2008;Wu and Nurse, 2009). The amounts of Sld2, Sld3, Sld7, Dpb11, Cdc45 and Dbf4 have been quantified in budding yeast, and they are well below the number of loaded Mcm2-7 helicases (Mantiero et al, 2011;.…”

Behavior of replication origins in Eukaryota – spatio-temporal dynamics of licensing and firing

“…In X. laevis egg extracts, a decrease in Cdc45 level reduced the efficiency of replication, 99 in S. pombe, its overexpression increased the early S phase replication. [100][101] The same correlation exists in mammalian cells, where Cdc45 is present at a relatively low level compared to the total number of licensed origins, and its increase resulted in a higher number of replisomes. 12 It was shown that in S. cerevisiae, late-firing ROs were directly dependent not only on Cdc45 levels, but also on Sld3, Sld7 or DDK subunits of Cdc7 and Dbf4.…”

Helicase Activation and Establishment of Replication Forks at Chromosomal Origins of Replication

“…The pre-RCs are assembled at all potential origins in G 1 phase, and formation of the pre-ICs determines the timing of origin firing in budding yeast because the replication proteins that assemble at this step are limited. Origins with a high affinity for such factors fire earlier in S phase (Patel et al 2008;Wu and Nurse 2009;Mantiero et al 2011;Tanaka et al 2011a). For details regarding the temporal regulation of replication, see Rhind and Gilbert (2013).…”