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. 2002 Jan 8;99(1):101-6.
doi: 10.1073/pnas.012578499. Epub 2001 Dec 26.

The B2 Element of the Saccharomyces Cerevisiae ARS1 Origin of Replication Requires Specific Sequences to Facilitate pre-RC Formation

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The B2 Element of the Saccharomyces Cerevisiae ARS1 Origin of Replication Requires Specific Sequences to Facilitate pre-RC Formation

Gwendolyn M Wilmes et al. Proc Natl Acad Sci U S A. .
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Abstract

The minimal requirements for a eukaryotic origin of replication are an initiator binding site and a region of helically unstable DNA [DNA unwinding element (DUE)]. Budding yeast origins consist of modular elements, and one of these elements, B2, has been proposed to act as a DUE. To test this hypothesis, we screened for sequences that function at the B2 element of ARS1. We found that the B2 element required A-rich sequences, but that the function of these identified sequences did not correlate with helical instability. Instead, the sequences that substituted fully for B2 function showed similarity to the ARS consensus sequence (ACS). The ACS is the binding site for the initiator origin recognition complex (ORC), but the selected sequences are not strong ORC binding sites in vitro. Nonfunctional B2 sequences show a corresponding loss in Mcm2-7p origin association. The function of these mutant sequences is rescued by Cdc6p overexpression. We propose that the B2 element requires specific sequences to bind a component of the pre-RC.

Figures

Figure 1
Figure 1
ARS1 B2 mutant sequences. (A) The B2 region of ARS1. A XhoI to PstI fragment of ARS1 with randomized bases at the 11-bp B2 element was cloned into the XhoI to PstI region of pARS1/785-92. Note that the strand referred to throughout this paper is the Lower strand in this figure, the A-rich strand of the B2 element, read from right to left 5′ to 3′. The A and B2 element ACS matches are shown as red arrows. (BE) The frequency of each nucleotide on the y axis is plotted against each of the 11 randomized positions on the x axis. (B) The library represents all 4 bases at all 11 positions. Twenty-five clones from the E. coli library were sequenced. (C) The B2 sequences are representative of the library as a whole. (D) The B2+ sequences are A-rich and weakly match up to the ACS starting at the second nucleotide. The ACS is aligned underneath the graph for comparison. (E) The B2+ sequences form a consensus that strongly matches the ACS if two-thirds of the sequences are shifted 1 base to the right. The B2+ sequences from D were examined to find the best match to the ACS within 1 base of the randomized 11 bases. The ACS is provided below for comparison.
Figure 2
Figure 2
Helical stability does not correlate with origin function of ARS1 B2 mutants. The helical stability of the mutant B2 element sequences was calculated as described in Materials and Methods. The helical stability was plotted against origin function for all B2 element mutants whose plasmid stability had been measured more than once, and a linear regression trend line and correlation value (R2) were assigned by excel (Microsoft).
Figure 3
Figure 3
Orc1-5p cannot bind representative B2+ sequences in vitro. DNase I protection assays were performed with wild-type Orc1-6p (lanes 1–3), or Orc1-5p (lanes 4–18). Orc1-6p protects the A and B1 elements of wild-type ARS1 DNA (lanes 1–3), whereas Orc1-5p protects the A element and the B2 element of wild-type ARS1 DNA (lanes 4–6), with characteristic hypersensitive sites in between. In contrast, Orc1-5p does not protect the B2 elements of the B2+ ARS1 DNAs (lanes7–18). Lanes 1, 4, 7, 10, 13, and 16 contain no protein. Lanes 2, 5, 8, 11, 14, and 17 contain 50 ng of protein. Lanes 3, 6, 9, 12, 15, and 18 contain 250 ng of protein. M1–M4 refer to the B2 mutants listed in Table 2.
Figure 4
Figure 4
MCM origin association correlates with B2 mutant origin function. Strains containing ARS1 mutations were arrested in the G1 phase of the cell cycle with α factor and assayed for MCM association by ChIP. PCR was performed on both the immunoprecipitated (IPed) DNA and the input DNA with primers to the origins ARS305 and ARS1, and the non-origin sequence URA3. Sample IPed PCR is shown above. The graph represents the quantitation of the (ARS1 IP/ARS1 input)/(ARS305 IP/ARS305 input) for four individual experiments. Origin function is plotted on the second axis. The A element mutation is ARS1/865–872 and does not have a plasmid loss rate because plasmids containing this origin cannot transform. M1–M9 refer to the B2 mutants listed in Table 2.
Figure 5
Figure 5
Overexpression of wild-type but not mutant Cdc6p rescues B2 mutant origins. (A) A wild-type plasmid (pARS1/785-92) is lost at a higher rate when wild-type Cdc6p is overexpressed, whereas the plasmid loss of a B2 mutant plasmid (pARS1/802–810) decreases to the wild-type level. Plasmids were transformed into strains containing wild-type or mutant CDC6 under control of the Gal1-10 promoter, and the cells grown in media containing 2% galactose. (B) A series of representative B2 mutant plasmids are improved by galactose-induced overexpression of Cdc6p. Control, no overexpression. Gal, galactose. M5–M10 refer to the B2 mutants listed in Table 2.

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