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. 2001 Sep 25;98(20):11152-7.
doi: 10.1073/pnas.191387498. Epub 2001 Sep 18.

In Vivo Interactions of Archaeal Cdc6/Orc1 and Minichromosome Maintenance Proteins With the Replication Origin

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Free PMC article

In Vivo Interactions of Archaeal Cdc6/Orc1 and Minichromosome Maintenance Proteins With the Replication Origin

F Matsunaga et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Although genome analyses have suggested parallels between archaeal and eukaryotic replication systems, little is known about the DNA replication mechanism in Archaea. By two-dimensional gel electrophoreses we positioned a replication origin (oriC) within 1 kb in the chromosomal DNA of Pyrococcus abyssi, an anaerobic hyperthermophile, and demonstrated that the oriC is physically linked to the cdc6 gene. Our chromatin immunoprecipitation assays indicated that P. abyssi Cdc6 and minichromosome maintenance (MCM) proteins bind preferentially to the oriC region in the exponentially growing cells. Whereas the oriC association of MCM was specifically inhibited by stopping DNA replication with puromycin treatment, Cdc6 protein stayed bound to the replication origin after de novo protein synthesis was inhibited. Our data suggest that archaeal and eukaryotic Cdc6 and MCM proteins function similarly in replication initiation and imply that an oriC association of MCM could be regulated by an unknown mechanism in Archaea.

Figures

Figure 1
Figure 1
Physical map of a P. abyssi chromosomal segment (sequence coordinates 118,000–128,000) carrying the predicted replication origin (12). Locations of several restriction sites (H, HindIII; N, NheI; E, EcoRI; X, XbaI), probes used for N/N 2D analyses are indicated. Fragments A (4.9-kb NheI fragment), B (5.7-kb EcoRI fragment), C (4.1-kb HindIII fragment), and D (5.6-kb XbaI fragment) correspond to those analyzed in Fig. 2. The central third of the each fragment [a bubble detection zone (18)] is indicated by gray shading. A ≈1-kb boxed region upstream of, and partially overlapping with, the P. abyssi cdc6 indicates the region containing an active replication origin as demonstrated in Fig. 2.
Figure 2
Figure 2
N/N 2D gel analyses of replication intermediates from the predicted replication origin region of P. abyssi. Replication intermediates were prepared from asynchronous P. abyssi cultures by an agarose plug method. A–D correspond to the analysis of restriction fragments A to D with the use of probe 1 (Fig. 1). (E) The expected pattern for fragments containing either one replication fork (Y arc) or two replication forks with an internal initiation site (bubble arc). The filled arrows indicate the bubble arcs observed for fragments B and C by probe 1 (similar results for fragments B and C were obtained when probe 2 was used). In addition to a well-defined Y arc, fragments A and D show weak signals (small arrows) detected by probe 1. These signals are consistent with an asymmetrically located replication origin within the central third of the fragments B and C. The mobilities of the dark spots on the line traced by linear fragments are consistent with impartial digests. (F) Structure of the oriC region with the identification of the two duplex unwinding elements (DUE) (19) and conserved nucleotide repeats (indicated by arrows) (12). Longer arrows indicate 34-bp repeats earlier unnoticed.
Figure 3
Figure 3
Chromatin association of P. abyssi Cdc6 (PAB2265) and MCM (PAB2373). (A) Expression patterns of P. abyssi Cdc6 and MCM were investigated by immunoblotting of 10% or 8%, respectively, SDS/PAGE gels after various times after inoculation. Puromycin was added after 4 h of growth. The cell densities of samples (107 cells per milliliter) are indicated below each lane. As whole-cell lysates were directly analyzed, the results should be considered semiquantitative. (B) Chromatin binding of P. abyssi Cdc6 and MCM was investigated by separating cell lysates into soluble (S) and chromatin-enriched (P) fractions. Where indicated, puromycin (200 μg/ml)-treated or stationary phase cells were used for assay. All samples were extracted from a comparable amount of cells. Protein amounts analyzed per lane are indicated below each lane in micrograms.
Figure 4
Figure 4
In vivo association of Cdc6 and MCM with the P. abyssi replication origin detected by formaldehyde cross-linking. (A) The locations of oriC and cdc6 in P. abyssi sequence coordinates, as well as five primer sets used for PCR amplification (a, 113,000; b, oriC (Left); c, oriC (Right); d, 133,000; control, 745,000) within a genome segment (110,000–135,000). (B) Specific association of Cdc6 and MCM proteins with chromatin was addressed with specific antisera and primer sets a–d, normalized against signals observed for the control locus 624 kb from the origin. 1/240, 1/960, and 1/3,840 of total precipitated DNA were used as PCR templates (loaded from left to right). Input and Mock refer to reactions performed with total sonicated input DNA and negative control reactions performed without antiserum, respectively. Analyses were performed with exponential-phase (with and without puromycin treatment) and stationary-phase cultures. (C) The relative abundance of each PCR product obtained under various conditions is indicated; 0.8 kb refers to data obtained with the use of an 800-nt-long oriC-specific PCR target (data not shown).
Figure 5
Figure 5
A schematic model of archaeal initiation complexes at the replication origin in replicating (exponential-phase culture) and nonreplicating (puromycin-treated samples) P. abyssi cells. The shaded box and arrow indicate the P. abyssi cdc6 gene and its transcriptional start in the vicinity of the mapped oriC. Cdc45 and Cdt1 missing in Archaea are essential for replication initiation in Eukarya. RC, replication complex.

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