Assembly of a Complex Containing Cdc45p, Replication Protein A, and Mcm2p at Replication Origins Controlled by S-Phase Cyclin-Dependent Kinases and Cdc7p-Dbf4p Kinase

Abstract

In Saccharomyces cerevisiae, replication origins are activated with characteristic timing during S phase. S-phase cyclin-dependent kinases (S-CDKs) and Cdc7p-Dbf4p kinase are required for origin activation throughout S phase. The activation of S-CDKs leads to association of Cdc45p with chromatin, raising the possibility that Cdc45p defines the assembly of a new complex at each origin. Here we show that both Cdc45p and replication protein A (RPA) bind to Mcm2p at the G 1 -S transition in an S-CDK-dependent manner. During S phase, Cdc45p associates with different replication origins at specific times. The origin associations of Cdc45p and RPA are mutually dependent, and both S-CDKs and Cdc7p-Dbf4p are required for efficient binding of Cdc45p to origins. These findings suggest that S-CDKs and Cdc7p-Dbf4p promote loading of Cdc45p and RPA onto a preformed prereplication complex at each origin with preprogrammed timing. The ARS1 association of Mcm2p, but not that of the origin recognition complex, is diminished by disruption of the B2 element of ARS1, a potential origin DNA-unwinding element. Cdc45p is required for recruiting DNA polymerase ␣ onto chromatin, and it associates with Mcm2p, RPA, and DNA polymerase only during S phase. These results suggest that the complex containing Cdc45p, RPA, and MCMs is involved in origin unwinding and assembly of replication forks at each origin.In eukaryotic cells, DNA replication initiates from multiple origins scattered along chromosomes, making duplication of large genomes within a single cell cycle possible. Every replication origin fires once and only once with a characteristic timing during the S phase. Although the cell-cycle control of DNA replication has been gradually unveiled, how DNA replication is initiated at chromosomal origins remains largely unknown (reviewed in references 16, 21, 46, and 57).Studies of Saccharomyces cerevisiae and other organisms have revealed that a prereplication complex (pre-RC) is assembled at each origin during late mitosis or early G 1 phase, the period when cyclin-dependent kinases (CDKs) are inactive (11,12,15,40,51,58,59). The origin recognition complex (ORC), Cdc6p, and the six minichromosome maintenance (MCM) proteins Mcm2p to -7p are present in the pre-RC (2,19,40,55,59,63). To start DNA synthesis, S-phase CDKs (S-CDKs) and Cdc7p-Dbf4p kinase need to be activated at the G 1 -S transition. Upon activation of these kinases, the pre-RC undergoes a transformation, and it is eventually converted to the post-RC and replication forks when initiation of DNA replication is completed (16,21,46,57). To understand how DNA replication is initiated at chromosomal origins, we sought to uncover how the complexes at origins change during initiation and how they interact with origins. Furthermore, we pursued the mechanisms by which the activating kinases are involved in this transition.In the well-characterized simian virus 40 (SV40) system, large T antigen is the origin recognition protein and also the helicase. Initiation of DNA replication from the...

Perpetuating the double helix: molecular machines at eukaryotic DNA replication origins

“…(73) Co-immunoprecipitation experiments have shown that Cdc45 interacts with components of the pre-RC like Mcm2, but also with RPA and DNA polymerases a and e, (74) suggesting that Cdc45 may serve as a bridge between the pre-RC and the proteins required for elongation. Indeed, Cdc45 association with the origins correlates with their time of activation and requires active CDKs and DDK.…”

“…Indeed, Cdc45 association with the origins correlates with their time of activation and requires active CDKs and DDK. (74,75) Its function is not limited to facilitating initiation because the controlled destruction of a degron-tagged CDC45 allele also affected elongation. (76) A recent study has suggested a role for XCdc45 as an auxiliary factor for the helicase activity of MCMs.…”

Analysis of Cdc6 function in the assembly of mammalian prereplication complexes

“…At least one of those functions is likely to be the loading of the Cdc45 protein onto the newly formed prereplication complex (35,47,48), although the precise mechanism of this aspect of cyclin E͞Cdk2 function remains to be elucidated.…”

Expression of MCM10 and TopBP1 is regulated by cell proliferation and UV irradiation via the E2F transcription factor

“…In the second step, Cdc7/Dbf4 kinase and S-phase cyclindependent kinases (S-Cdk) activate the pre-RC and trigger DNA replication by loading Cdc45 onto each origin with programmed timing (Bell and Dutta, 2002). Cdc45 facilitates assembly of the replication machinery by recruiting replication protein A and DNA polymerase a (Mimura and Takisawa, 1998;Zou and Stillman, 2000). In addition to S-Cdk and Cdc7, other factors, such as MCM10 and TopBP1, have also been shown to recruit Cdc45 (Van Hatten et al, 2002;Wohlschlegel et al, 2002).…”