The origin recognition complex interacts with a bipartite DNA binding site within yeast replicators.

Abstract

Replicators are genetically defined elements within chromosomes that determine the location of origins of DNA replication. In the yeast Saccharomyces cerevisiae, the ARSI replicator contains multiple functional DNA elements: an essential A element and three important B elements-Bi, B2, and B3. Functionally similar A, Bi, and B2 elements are also present in the ARS307 replicator. Eukaryotic cells duplicate their genetic information for the next generation during the S phase of the cell division cycle. In general, two steps are required for a cell to duplicate its genome. First, a multiprotein complex is assembled at specific sites within the chromosome where DNA replication starts (the replication origins). Second, the enzymatic machinery that replicates the DNA is activated and replication forks move bidirectionally to synthesize the new DNA chains (1). The replication process is highly regulated at the initiation step. Studies in bacteria and with viruses that infect mammalian cells support the replicon model (2), which posits that DNA replication initiates at a specific site (origin) and this initiation is controlled by a cis-acting DNA sequence called the replicator (1-3). A replicator-specific DNA binding protein called the initiator facilitates the assembly of the enzymatic machinery at the replication origin to allow initiation of DNA replication to proceed.The nature of replicators and the proteins that are involved in the initiation of DNA replication are the keys to understanding the mechanism and regulation of initiation of eukaryotic DNA replication. Autonomous replicating sequences (ARSs) were identified in the yeast Saccharomyces cerevisiae by their ability to promote the maintenance of extrachromosomal DNA (4-7). Subsequently, both replicators and origins of DNA replication in the chromosome were found to colocalize with a subset of these ARS elements (7). Fine structure analysis of the ARS1 and ARS307 replicators demonstrated that they contain multiple short functional elements (8)(9)(10) (7,8). The B3 element is found in some but not all of the ARSs and it functions in an orientation-and position-independent manner to enhance origin utilization (12, 13).A multisubunit origin recognition complex (ORC) was identified as a candidate initiator protein using the DNase I footprint method and was shown to bind toARSJ and all other ARS sequences in an ATP-dependent manner (14,15). ORC contains six subunits of 120, 72, 62, 56, 53, and 50 kDa. Genes encoding all six subunits have been cloned and found to be essential for cell viability (S. P. Bell, R. Kobayashi, and B.S., unpublished data; see refs. 15-18), Further genetic analyses suggest that ORC plays an important role in DNA replication (reviewed in ref. 7). The ORC DNase I protection pattern at ARS sequences extends over a 50-bp region that includes the A and Bi elements (9,14). In addition, several DNase I-hypersensitive sites are induced in the DNA. These hypersensitive sites are found at 10-bp intervals, suggesting that the DNA may wrap aro...

Human Origin Recognition Complex Binds Preferentially to G-quadruplex-preferable RNA and Single-stranded DNA

“…We confirmed that this purified ORC was functionally active because it could partially rescue DNA replication in the ORCdepleted, Xenopus egg extracts (data not shown), as shown previously (11). Biotin-labeled, ϳ110-bp dsDNA containing (GGGTT) 8 or a 40-bp AT-rich element was used as a probe for EMSA, and binding specificity was investigated by adding unlabeled competitors into the binding reaction (see Table 1 for their sequences). First, we compared two competitors, AT-rich and (GGGTT) 8 dsDNA (ϳ40 bp) to evaluate the conditions for EMSA.…”

“…(GGGTT) 8 ssDNA, but not (AACCC) 8 ssDNA, inhibited DNA binding of ORC more efficiently than (GGGTT) 8 dsDNA (Fig. 1D); a 10-fold molar excess of (GGGTT) 8 ssDNA almost completely inhibited ORC binding to an AT-rich dsDNA probe, but (GGGTT/CCCAA) 8 dsDNA or (AACCC) 8 ssDNA was less effective. (GGGTT) 8 inhibited as efficiently as AT-rich dsDNA (Fig.…”

“…For the biotinlabeled dsDNA probes (GGGTT/CCCAA or A/T-rich), the corresponding repeated (or A/T-rich) DNA (40 bp) was cloned into the SacI-KpnI sites in pBluescript SK(Ϫ) (Agilent Technologies) and amplified by PCR with biotin-labeled T7/T3 primers. For the biotin-labeled, (GGGUU) 8 -containing RNA probe (58 nucleotides), the plasmid as above was linearized and used for in vitro transcription with T7 RNA polymerase. The RNA was purified by denatured polyacrylamide gel electrophoresis and biotinylated at the 3Ј-end with the RNA 3Ј-end biotinylation kit (Thermo Scientific).…”

“…In contrast, the mechanism for ORC to recognize the origins seems different in eukaryotic species. In S. cerevisiae, ORC can bind sequence specifically to the consensus sequence within autonomously replicating sequences (3,8). In another yeast, Schizosaccharomyces pombe, ORC can recognize the AT-tracts present in autonomously replicating sequences; the AT-hook motif of the ORC4 subunit is responsible for this recognition (9).…”

The architecture of the DNA replication origin recognition complex in Saccharomyces cerevisiae

“…I n Saccharomyces cerevisiae origins of DNA replication contain conserved A, B1, and B2 elements, where the A element and part of B1 define the binding sequence for the origin recognition complex (ORC) (1)(2)(3). ORC binds to origin DNA in an ATPdependent manner and recruits other essential proteins, such as the initiation factors Cdc6, Cdt1, and the presumptive DNA helicase MCM, to the autonomously replicating sequence (ARS) to form a prereplicative complex (pre-RC) before the initiation of DNA replication that occurs in S phase (4)(5)(6)(7).…”