Modulation of DNA Synthesis in Saccharomyces cerevisiae Nuclear Extract by DNA Polymerases and the Origin Recognition Complex

Abstract

We have analyzed the modulation of DNA synthesis on a supercoiled plasmid DNA template by DNA polymerases (pol), minichromosome maintenance protein complex (Mcm), topoisomerases, and the origin recognition complex (ORC) using an in vitro assay system. Antisera specific against the four-subunit pol ␣, the catalytic subunit of pol ␦, and the Mcm467 complex each inhibited DNA synthesis. However, DNA synthesis in this system appeared to be independent of pol⑀. Consequently, DNA synthesis in the in vitro system appeared to depend only on two polymerases, ␣ and ␦, as well as the Mcm467 DNA helicase. This system requires supercoiled plasmid DNA template and DNA synthesis absolutely required DNA topoisomerase I. In addition, we also report here a novel finding that purified recombinant six subunit ORC significantly stimulated the DNA synthesis on a supercoiled plasmid DNA template containing an autonomously replicating sequence, ARS1.Studies on the replication of genomes of bacteriophages, plasmids, viruses, and prokaryotic and eukaryotic cells have established general mechanisms of DNA replication (1). The eukaryotic origins of DNA replication were first discovered in the budding yeast, Saccharomyces cerevisiae, and were named autonomously replicating sequences (ARS) 1 (2-4). Many yeast ARS elements have been characterized in detail, which has allowed the discovery of many cis-acting replication proteins and regulators of DNA replication (5, 6). The discovery by Bell and Stillman (7,8) that the in vivo initiation of DNA replication in S. cerevisiae requires a six subunit origin recognition complex (ORC) is a major advancement in understanding the mechanism of eukaryotic DNA replication. In the G 1 phase of the cell cycle, Cdc6p, Cdt1p, minichromosome maintenance (Mcm), and Cdc45p proteins bind sequentially to DNA to form a prereplication complex (pre-RC) (9 -11). At the G 1 /S boundary, S phase-specific cyclin-dependent protein kinases (Cdks) and particularly the Cdc7/Dbf4 kinase transform the pre-RC into an active replication complex (10 -12). Finally, the initiation coincides with the association of the polymerase ␣/primase (pol ␣/primase) complex to the RPA-coated unwound origins (10). S and M phase-specific Cdks block the re-binding of Mcm to the chromatin and prevent a new round of initiations until mitosis (13).The Mcm protein family that is required for DNA replication in eukaryotes consists of six proteins (Mcm 2-7) with highly conserved amino acids between the six polypeptides. All of these proteins are essential for the cell viability (13, 14). The Mcm467 proteins form a relatively stable core complex and other Mcm proteins are loosely associated to it (14 -17). It has been demonstrated that a DNA helicase activity is associated with the Mcm467 complex, suggesting that this complex is involved in the initiation of DNA replication as a DNA unwinding enzyme (18,19). Furthermore, Mcm467 remains associated with the replication fork during the elongation step of DNA replication (20). Among a number of candidate D...

Homeotic proteins participate in the function of human-DNA replication origins

“…The lamin B2 origin sequence is characterized by an intrinsic tendency to form such unusual structures (47) and contains a bent DNA sequence between nt 3923 and 3928. Furthermore, topological status is clearly critical for origin recognition by ORC in yeast and Drosophila origins (48,49), while topo I and II bind precise sites in the lamin B2 pre-RC area in precise moments of the cell cycle, topo I being essential for synthesis initiation (44,50). In this context, it is particularly important to observe that both topos precisely recognize in vitro the same origin nucleotides cleaved in vivo .…”

A novel role for RAD54: this host protein modulates geminiviral DNA replication

“…The yeast cells were synchronized at the G 1 or S phase using α‐factor, and the spheroplasts were obtained by following the procedure described by Verdier et al (46). The NE S s were prepared as described previously (46, 47). The plasmids 2A and OriM‐2A were acid‐phenol extracted to selectively remove the nicked and linear DNA, and >95% of the purified plasmids were in the supercoiled form.…”

“…The assays were essentially performed as described by Mitkova et al (47) with slight modifications. Typically, 25 μl of a reaction mixture in a buffer (20 mM HEPES/NaOH, pH 7.6; 5 mM MgCl 2 ; 0.3 mM EDTA‐NaOH, pH 7.6; and 1 mM DTT) contained 300 ng supercoiled plasmid DNA, 40 mM creatine phosphate, 0.125 mg/ml creatine phosphokinase, 4 mM ATP, 70 μM of each rNTP, 100 μM of each dNTP, 0.125 μCi of [α‐ 32 P]dTTP or [α‐ 32 P]dCTP, and the indicated proteins.…”

Functional interactions of DNA topoisomerases with a human replication origin

“…The regulation of DNA replication of eukaryotic organisms is mediated by the cell‐cycle‐dependent assembly and reorganization of specific multiprotein complexes on the origins of DNA replication. DNA topoisomerases are required for the processes of activation of the ori C of Escherichia coli (Kornberg, 1984) and of the origins of SV40 (Halmer et al , 1998), BPV (Hu et al , 2006) and EBV genomes (Kawanishi, 1993); furthermore, in vitro DNA synthesis with a Saccharomyces cerevisiae system requires negatively supercoiled DNA and the action of DNA topoisomerase I (topo I) (Mitkova et al , 2005), and negatively supercoiled DNA is also required for the binding of the Drosophila ORC (Remus et al , 2004). Thus, the modifications of the specific protein–DNA interactions occurring at human replication origins may entail DNA topoisomerase‐induced modulations of the topological state of the origin DNA.…”