Summary Elevated hippocampal activation is observed in conditions that confer risk for Alzheimer's disease, including amnestic mild cognitive impairment (aMCI). Studies in relevant animal models have indicated that over-activity in selective hippocampal circuits contributes to cognitive impairment. Here we tested the effect of reducing hippocampal activation in aMCI. Under placebo treatment, hippocampal activation in the dentate gyrus/CA3 was elevated in aMCI patients compared to a healthy control group. By using a low dose of the antiepileptic levetiracetam hippocampal activation in aMCI was reduced to a level that did not differ from the control group. Compared to aMCI memory performance under placebo, performance in the scanning task was significantly improved under drug treatment. Contrary to the view that greater hippocampal activation might serve a beneficial function, these results support the view that increased hippocampal activation in aMCI is a dysfunctional condition and that targeting excess hippocampal activity has therapeutic potential.

During pre-replication complex (pre-RC) formation, origin recognition complex (ORC), Cdc6, and Cdt1 cooperatively load the 6-subunit mini chromosome maintenance (MCM2-7) complex onto DNA. Loading of MCM2-7 is a prerequisite for DNA licensing that restricts DNA replication to once per cell cycle. During S phase MCM2-7 functions as part of the replicative helicase but within the pre-RC MCM2-7 is inactive. The organization of replicative DNA helicases before and after loading onto DNA has been studied in bacteria and viruses but not eukaryotes and is of major importance for understanding the MCM2-7 loading mechanism and replisome assembly. Lack of an efficient reconstituted pre-RC system has hindered the detailed mechanistic and structural analysis of MCM2-7 loading for a long time. We have reconstituted Saccharomyces cerevisiae pre-RC formation with purified proteins and showed efficient loading of MCM2-7 onto origin DNA in vitro. MCM2-7 loading was found to be dependent on the presence of all pre-RC proteins, origin DNA, and ATP hydrolysis. The quaternary structure of MCM2-7 changes during pre-RC formation: MCM2-7 before loading is a single hexamer in solution but is transformed into a double-hexamer during pre-RC formation. Using electron microscopy (EM), we observed that loaded MCM2-7 encircles DNA. The loaded MCM2-7 complex can slide on DNA, and sliding is not directional. Our results provide key insights into mechanisms of pre-RC formation and have important implications for understanding the role of the MCM2-7 in establishment of bidirectional replication forks.helicase ͉ initiation ͉ mini chromosome maintenance ͉ ORC ͉ pre-RC

Importin ␣ plays a pivotal role in the classical nuclear protein import pathway. Importin ␣ shuttles between nucleus and cytoplasm, binds nuclear localization signal-bearing proteins, and functions as an adapter to access the importin ␤-dependent import pathway. In contrast to what is found for importin ␤, several isoforms of importin ␣, which can be grouped into three subfamilies, exist in higher eucaryotes. We describe here a novel member of the human family, importin ␣7. To analyze specific functions of the distinct importin ␣ proteins, we recombinantly expressed and purified five human importin ␣'s along with importin ␣ from Xenopus and Saccharomyces cerevisiae. Binding affinity studies showed that all importin ␣ proteins from humans or Xenopus bind their import receptor (importin ␤) and their export receptor (CAS) with only marginal differences. Using an in vitro import assay based on permeabilized HeLa cells, we compared the import substrate specificities of the various importin ␣ proteins. When the substrates were tested singly, only the import of RCC1 showed a strong preference for one family member, importin ␣3, whereas most of the other substrates were imported by all importin ␣ proteins with similar efficiencies. However, strikingly different substrate preferences of the various importin ␣ proteins were revealed when two substrates were offered simultaneously.

Binding of Cdc6p to the Origin-Recognition-Complex (ORC) is a key step in the assembly of a prereplication complex (pre-RC) at origins of DNA replication. ORC recognizes specific origin DNA sequences in an ATP-dependent manner. Here we demonstrate cooperative binding of Saccharomyces cerevisiae Cdc6p to ORC on DNA in an ATP-dependent manner, inducing a change in the pattern of origin binding that requires the Orc1p ATPase. The reaction is blocked by specific origin mutations that do not interfere with the interaction between ORC and DNA. Single particle reconstruction of electron microscopic images shows that the ORC-Cdc6p complex forms a ringshaped structure with dimensions similar to the ring-shaped MCM helicase. The ORC-Cdc6p structure is predicted to contain six AAA+ subunits, analogous to other ATP-dependent protein machines. We suggest that Cdc6p and origin DNA activate a molecular switch in ORC that contributes to pre-RC assembly.Pre-replication complex (pre-RC) assembly at origins of DNA replication is essential to license chromosomes before initiation of DNA synthesis occurs during S phase of the cell division cycle 1-3. The Origin-Recognition-Complex (ORC), a six-subunit, ATP-dependent DNA binding protein binds to specific DNA sequences at origins of DNA replication and is the foundation for pre-RC assembly 4-6. The Orc1p and Orc5p subunits are known to interact with ATP, however only the interaction between the Orc1p subunit and ATP is required for DNA binding and is essential in yeast. Once ORC is bound to origin DNA, the ORC ATPase is reduced or blocked7 -9. Binding of Cdc6p to ORC is a key step in the assembly of the pre-RC [10][11][12][13][14][15][16][17][18][19][20] . In particular, the levels and activity of Cdc6 regulate the frequency with which any given origin is utilized during the cell cycle 10,11,21 .In yeast origins of DNA replication contain specific DNA elements, A, B1 and B2, not all of which are conserved at the nucleotide sequence level 22 . The A-element is essential and represents the principal binding site for the ORC. The B1 element is partially involved in ORC-DNA interaction and B2 mutants are impaired in loading Mini-Chromosome Maintenance (MCM) proteins on chromatin 5, 6 , 22 -24. The pre-RC was first described using an in vivo DNase I footprinting method 25 . During the G2 phase of the cell cycle, the DNase I cleavage pattern at origins was consistent with ORC bound to DNA (i.e. protection over the A and B1 elements). In G1 phase, however, the pattern changed and a prominent hypersensitive site within B1 disappeared and the regions between B1 and B2 and the B2 element itself were protected. Since this change corresponded with chromatin binding of the MCM proteins and DNA licensing it has been assumed that MCM binding to DNA was responsible for the larger,