During replication the genome must be duplicated accurately and exactly once per cell cycle to guarantee genetic homeostasis. The first step, called replication initiation, regulates when exactly and where in the genome DNA replication occurs. These regulatory steps and the essential proteins involved in this process are well- characterized in yeast. For the tight control of replication, initiation is divided into a two- step process that separates the loading of the replicative helicase in G1 phase from its activation in S phase of the cell cycle. During the first step, origin licensing, the Mcm2-7 helicase is loaded onto origin DNA and thereby forms the pre-replicative complex (pre- RC). Origin firing, the second step of replication initiation, mediates the conversion of the inactive pre-RC into the active Cdc45-Mcm2-7-GINS (CMG) helicase. The major regulatory step in this conversion is the assembly of an intermediate complex, the so- called pre-initiation complex (pre-IC). During pre-IC assembly the essential helicase components Cdc45 and GINS are recruited to the Mcm2-7 helicase. In yeast, Cdc45 recruitment to the helicase is mediated by the Sld3-Sld7 complex (metazoan Treslin- MTBP) and GINS is delivered as part of the so-called pre-loading complex (pre-LC). Although most of the core replication factors are conserved from yeast to humans, the molecular details of Cdc45 and GINS loading onto the pre-RC are not yet known in metazoans. Here we demonstrate that the essential firing factor TopBP1 interacts with GINS through two individual binding surfaces during S phase. TopBP1 is a multi-BRCT scaffold protein that fulfils multiple functions in metazoan cells. In particular, TopBP1 plays dual roles in replication origin firing and in the response to DNA damage. Our findings provide molecular details on how TopBP1 contributes to helicase activation during origin firing.

In this thesis we show that the previously described GINS interaction (GINI) domain of TopBP1 is highly conserved between metazoans. By protein sequence alignment between metazoan species, we narrow down the GINI domain to a conserved region between TopBP1 BRCT3 and BRCT4. Systematical mutations of this domain demonstrate that the GINI region is required for GINS binding in vitro but has only a moderate effect on replication in Xenopus egg extract. Structural analysis suggests that the GINI domain forms a short α helix. By integration of a mutation that breaks this helix we show that the helix provides a binding surface for GINS. Furthermore, we verify the corresponding binding residues in the distal part of the A-domain of the GINS subunit Psf1. Interestingly, all GINI mutants are incapable to bind to GINS in vitro but still support replication in egg extract to relatively high amounts. This controversial observation suggests that a second GINS binding motif is present in TopBP1, compensating for the loss of the GINI domain function. Indeed, we identify a second GINS interaction surface in the BRCT4 domain of TopBP1. This TopBP1 BRCT4 domain binds to the base of the Psf1 A-domain and to the linker between the A- and the B-domain. Functional analysis demonstrates that simultaneous mutations in both GINS interaction sites in TopBP1 lead to severe defects of replication in Xenopus egg extract, suggesting a functional cooperation between the two GINS binding surfaces in TopBP1.

Within the active replisome the DNA polymerase ε (Polε) associates with the CMG helicase whereas TopBP1, Treslin and MTBP leave the complex before DNA synthesis starts. Our structural data suggests that simultaneous binding of GINS to TopBP1 BRCT4 and to the Polε subunit PolE2 is mutually exclusive. Therefore, we propose a model that once TopBP1 and GINS are associated with the helicase the B-domain of GINS Psf1 undergoes a conformational change that favors PolE2 binding over BRCT4 binding. We hypothesize that low-affinity GINS binding to the only remaining GINS interaction surface in TopBP1, induces TopBP1 ejection, and allows recycling of this limiting firing factor.