Deletion of the cruciform binding domain in CBP/14-3-3 displays reduced origin binding and initiation of DNA replication in budding yeast

BACKGROUND: Initiation of eukaryotic DNA replication involves many protein-protein and protein-DNA interactions. We have previously shown that 14-3-3 proteins bind cruciform DNA and associate with mammalian and yeast replication origins in a cell cycle dependent manner. RESULTS: By expressing the human 14-3-3ε, as the sole member of 14-3-3 proteins family in Saccharomyces cerevisiae, we show that 14-3-3ε complements the S. cerevisiae Bmh1/Bmh2 double knockout, conserves its cruciform binding activity, and associates in vivo with the yeast replication origins ARS307. Deletion of the α5-helix, the potential cruciform binding domain of 14-3-3, decreased the cruciform binding activity of the protein as well as its association with the yeast replication origins ARS307 and ARS1. Furthermore, the mutant cells had a reduced ability to stably maintain plasmids bearing one or multiple origins. CONCLUSION: 14-3-3, a cruciform DNA binding protein, associates with yeast origins of replication and functions as an initiator of DNA replication, presumably through binding to cruciform DNA forming at yeast replicators

The Effect of Regulatory Sequence Elements upon the Initiation of DNA Replication of the Minute Virus of Mice

AbstractThe minute virus of mice (MVM) genome is a linear single-stranded length of approximately 5000 nucleotides of DNA with unique terminal palindromic sequences at both ends. The left (3′) hairpin is used to prime the initiation of DNA synthesis on parental single-strand DNA while the right (5′) hairpin or stem-plus-arms structure can also prime the initiation of DNA synthesis during synthesis of dimer and higher oligomers as well as synthesis of progeny single strands. Previous studies have shown that if viral duplex DNA was input into anin vitroDNA replication system using extracts from uninfected HeLa cells, the 5′ end of the molecule was able to form a hairpin and initiate DNA synthesis by DNA polymerase δ (Cossonset al.(1996),Virology216, 258–264). In this study, the effect of the deletion of knowncis-acting genetic elements upon the initiation of DNA replication was studied using a series of MVM mutants with deletions within the 5′ terminal region. Mutants containing deletions of elements A (nucleotides 4489–4636), B (nucleotides 4636–4695), and either one or both of the 65-bp repeats (nucleotides 4720–4785 and 4785–4849) were used as template in thein vitroDNA replication system. When element A was deleted, the efficiency of initiation decreased significantly. Subsequent removal of element B, leaving just the two 65-bp repeats, restored levels of initiation back to those seen in the wild-type genome. In the absence of either A or B both 65-bp repeats were necessary for efficient initiation, and removal of one of these repeats caused a decrease in efficiency. Thus, element B appeared to have a negative regulatory effect (in the absence of element A), and element A appeared to have a positive regulatory effect, at least in the presence of element B. These data demonstrate, for the first time, a complex interaction between thesecis-acting regulatory elements which can function as both positive or negative regulators in the initiation of MVM DNA replication

Increased origin activity in transformed versus normal cells: identification of novel protein players involved in DNA replication and cellular transformation

Using libraries of replication origins generated previously, we identified three clones that supported the autonomous replication of their respective plasmids in transformed, but not in normal cells. Assessment of their in vivo replication activity by in situ chromosomal DNA replication assays revealed that the chromosomal loci corresponding to these clones coincided with chromosomal replication origins in all cell lines, which were more active by 2–3-fold in the transformed by comparison to the normal cells. Evaluation of pre-replication complex (pre-RC) protein abundance at these origins in transformed and normal cells by chromatin immunoprecipitation assays, using anti-ORC2, -cdc6 and -cdt1 antibodies, showed that they were bound by these pre-RC proteins in all cell lines, but a 2–3-fold higher abundance was observed in the transformed by comparison to the normal cells. Electrophoretic mobility shift assays (EMSAs) performed on the most efficiently replicating clone, using nuclear extracts from the transformed and normal cells, revealed the presence of a DNA replication complex in transformed cells, which was barely detectable in normal cells. Subsequent supershift EMSAs suggested the presence of transformation-specific complexes. Mass spectrometric analysis of these complexes revealed potential new protein players involved in DNA replication that appear to correlate with cellular transformation

Transient dsDNA breaks during pre-replication complex assembly

Initiation of DNA replication involves the ordered assembly of the multi-protein pre-replicative complex (pre-RC) during G1 phase. Previously, DNA topoisomerase II (topo II) was shown to associate with the DNA replication origin located in the lamin B2 gene locus in a cell-cycle-modulated manner. Here we report that activation of both the early-firing lamin B2 and the late-firing hOrs8 human replication origins involves DNA topo II-dependent, transient, site-specific dsDNA-break formation. Topo IIβ in complex with the DNA repair protein Ku associates in vivo and in vitro with the pre-RC region, introducing dsDNA breaks in a biphasic manner, during early and mid-G1 phase. Inhibition of topo II activity interferes with the pre-RC assembly resulting in prolonged G1 phase. The data mechanistically link DNA topo IIβ-dependent dsDNA breaks and the components of the DNA repair machinery with the initiation of DNA replication and suggest an important role for DNA topology in origin activation

Cdc6 expression represses E-cadherin transcription and activates adjacent replication origins

The Cdc6 replication licensing factor acts as a molecular switch at the E-cadherin locus, leading to E-cadherin transcriptional repression and local activation of replication

DNA synthesis in a purine auxotrophic mutant of Chinese hamster cells

Note:When V79 pur 1 cells are maintained in the absence of purines, the cells stop growing but remain viable for up to 48 h. Within the first 6 h after purine deprivation DNA synthesis is inhibited and the cells are arrested in S phase. The purine deoxyribonucleoside triphosphate (dNTP} pools decrease in size and the pyrimidine (dNTP) pools expand. Restoration of purine nucleotides into the medium results in normal rates of DNA synthesis both in vivo and in vitro. Kinetic analyses of DNA synthesis in samples pulsed for 1 min and chased for 1 h show a delay in maturation of newly replicated Okazaki-type pieces to full size DNA. DNA fiber autoradiography reveals a retardation in the rate of replication fork movement but no apparent inhibition of initiation in active clusters of replication units. The results indicate that purine deprivation upsets the intracellular dNTP pool balance and inhibits the elongation of nascent DNA chains.Quand les cellules V79 pur 1 sont maintenues dans un milieu sans purines, elles arrêtent de pousser mais restent vivantes jusqu’à 48 h. Dans les premières 6 h qui suivent la privation des purines la synthèse de l'ADN est inhibée et les cellules sont arrêtées dans la phase S du cycle cellulaire. La grandeur des 'pools' des déoxynucleosides triphosphates (dNTP) des purines diminue et celle des pyrimidines augmente. Le taux normal de la synthèse d'ADN résulte de la restauration des nucléotides des purines dans le milieu aussi bien in vivo qu'in vitro. Une analyse kinésique de la synthèse de l'ADN montre un délai dans la maturation des fragments du type Okazaki nouvellement synthétisés en longues chaines d'ADN. L'autoradiographie des fibres d'ADN révèle un retardement du mouvement de la fourche de réplication. Par contre cette même technique ne met pas en évidence une inhibition d'initiation dans les groupes d'unités de réplication qui sont actifs. Les résultats indiquent que la privation des purines renverse la balance des 'pools' des dNTPs cellulaires et inhibe l'élongation des chaines d'ADN nouvellement synthétisées