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A DNA sequence element that advances replication origin activation time in Saccharomyces cerevisiae.

Pohl TJ, Kolor K, Fangman WL, Brewer BJ, Raghuraman MK - G3 (Bethesda) (2013)

Bottom Line: Eukaryotic origins of DNA replication undergo activation at various times in S-phase, allowing the genome to be duplicated in a temporally staggered fashion.Currently, there are two examples of DNA sequences that are known to advance origin activation time, centromeres and forkhead transcription factor binding sites.By combining deletion and linker scanning mutational analysis with two-dimensional gel electrophoresis to measure fork direction in the context of a two-origin plasmid, we have identified and characterized a 19- to 23-bp and a larger 584-bp DNA sequence that are capable of advancing origin activation time.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Cellular Biology Program, University of Washington, Seattle, Washington 98195.

ABSTRACT
Eukaryotic origins of DNA replication undergo activation at various times in S-phase, allowing the genome to be duplicated in a temporally staggered fashion. In the budding yeast Saccharomyces cerevisiae, the activation times of individual origins are not intrinsic to those origins but are instead governed by surrounding sequences. Currently, there are two examples of DNA sequences that are known to advance origin activation time, centromeres and forkhead transcription factor binding sites. By combining deletion and linker scanning mutational analysis with two-dimensional gel electrophoresis to measure fork direction in the context of a two-origin plasmid, we have identified and characterized a 19- to 23-bp and a larger 584-bp DNA sequence that are capable of advancing origin activation time.

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Related in: MedlinePlus

Determining distances over which the bias determinant can function. (A-D) Fork direction 2-D gel analysis on pN&S plasmids that have various lengths of sequence inserted within the NcoI-SmaI fragment, between the bias determinant and ARS1S. The amount of sequence that was inserted for each construct is indicated above each image. Percent initiation at ARS1S is indicated in the lower left corners. (E) Replication indices for chromosomal URA3 (pink) and ARS510 (blue) in wild-type (WT) cells harboring the bias determinant at its native location (closed diamonds) and in cells lacking the bias determinant (open diamonds) are plotted. ARS607 (black dashed arrow) and R11 (black dotted arrow) were used as early and late timing standards, respectively. In the WT strain, URA3 and ARS510 had replication indices of 0.73 and 0.4, respectively. In the strain lacking the bias determinant (YTP38), URA3 had a replication index of 0.66, whereas the replication index for ARS510 was 0.34.
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fig5: Determining distances over which the bias determinant can function. (A-D) Fork direction 2-D gel analysis on pN&S plasmids that have various lengths of sequence inserted within the NcoI-SmaI fragment, between the bias determinant and ARS1S. The amount of sequence that was inserted for each construct is indicated above each image. Percent initiation at ARS1S is indicated in the lower left corners. (E) Replication indices for chromosomal URA3 (pink) and ARS510 (blue) in wild-type (WT) cells harboring the bias determinant at its native location (closed diamonds) and in cells lacking the bias determinant (open diamonds) are plotted. ARS607 (black dashed arrow) and R11 (black dotted arrow) were used as early and late timing standards, respectively. In the WT strain, URA3 and ARS510 had replication indices of 0.73 and 0.4, respectively. In the strain lacking the bias determinant (YTP38), URA3 had a replication index of 0.66, whereas the replication index for ARS510 was 0.34.

Mentions: Density shift experiments on the strains analyzed in Figure 4B and Figure 5E were respectively performed in strains RM14-3a and KK14-3a (a URA+ version of RM14-3a) as previously described (McCarroll and Fangman 1988). The data were analyzed using an InstantImager or phosphorimager (Packard or BioRad, respectively). The comparative hybridization experiments were performed essentially as described (Friedman et al. 1995). Strain 3xRZeoDIR containing pN&Sdir or pN&SDdir (described in the section Results) was incubated for 1.25 generations in α-factor. Upon arrest in the G1 phase, an uninduced sample was taken and galactose was added to 2%. Excision of the ARS-less cassette was allowed to proceed for four hours. Glucose was then added to 2% and the cells were released from α-factor arrest at 37°, the restrictive temperature for cdc7ts. Upon cdc7 arrest, a sample was taken, and the cells were returned to 23° to allow entry into S phase. Samples were taken at 4-min intervals throughout S (60 min). The DNA was prepared by the “Smash and Grab” procedure (Hoffman and Winston 1987) and digested with BamHI and EagI. The samples were run on a 0.7% agarose gel, blotted onto Hybond (Amersham) nylon membrane, and hybridized to a BamHI-EagI fragment of pBR322 labeled with 32P. Quantification of the blots was performed on a PhosphorImager (Molecular Dynamics) using ImageQuant software.


A DNA sequence element that advances replication origin activation time in Saccharomyces cerevisiae.

Pohl TJ, Kolor K, Fangman WL, Brewer BJ, Raghuraman MK - G3 (Bethesda) (2013)

Determining distances over which the bias determinant can function. (A-D) Fork direction 2-D gel analysis on pN&S plasmids that have various lengths of sequence inserted within the NcoI-SmaI fragment, between the bias determinant and ARS1S. The amount of sequence that was inserted for each construct is indicated above each image. Percent initiation at ARS1S is indicated in the lower left corners. (E) Replication indices for chromosomal URA3 (pink) and ARS510 (blue) in wild-type (WT) cells harboring the bias determinant at its native location (closed diamonds) and in cells lacking the bias determinant (open diamonds) are plotted. ARS607 (black dashed arrow) and R11 (black dotted arrow) were used as early and late timing standards, respectively. In the WT strain, URA3 and ARS510 had replication indices of 0.73 and 0.4, respectively. In the strain lacking the bias determinant (YTP38), URA3 had a replication index of 0.66, whereas the replication index for ARS510 was 0.34.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3815058&req=5

fig5: Determining distances over which the bias determinant can function. (A-D) Fork direction 2-D gel analysis on pN&S plasmids that have various lengths of sequence inserted within the NcoI-SmaI fragment, between the bias determinant and ARS1S. The amount of sequence that was inserted for each construct is indicated above each image. Percent initiation at ARS1S is indicated in the lower left corners. (E) Replication indices for chromosomal URA3 (pink) and ARS510 (blue) in wild-type (WT) cells harboring the bias determinant at its native location (closed diamonds) and in cells lacking the bias determinant (open diamonds) are plotted. ARS607 (black dashed arrow) and R11 (black dotted arrow) were used as early and late timing standards, respectively. In the WT strain, URA3 and ARS510 had replication indices of 0.73 and 0.4, respectively. In the strain lacking the bias determinant (YTP38), URA3 had a replication index of 0.66, whereas the replication index for ARS510 was 0.34.
Mentions: Density shift experiments on the strains analyzed in Figure 4B and Figure 5E were respectively performed in strains RM14-3a and KK14-3a (a URA+ version of RM14-3a) as previously described (McCarroll and Fangman 1988). The data were analyzed using an InstantImager or phosphorimager (Packard or BioRad, respectively). The comparative hybridization experiments were performed essentially as described (Friedman et al. 1995). Strain 3xRZeoDIR containing pN&Sdir or pN&SDdir (described in the section Results) was incubated for 1.25 generations in α-factor. Upon arrest in the G1 phase, an uninduced sample was taken and galactose was added to 2%. Excision of the ARS-less cassette was allowed to proceed for four hours. Glucose was then added to 2% and the cells were released from α-factor arrest at 37°, the restrictive temperature for cdc7ts. Upon cdc7 arrest, a sample was taken, and the cells were returned to 23° to allow entry into S phase. Samples were taken at 4-min intervals throughout S (60 min). The DNA was prepared by the “Smash and Grab” procedure (Hoffman and Winston 1987) and digested with BamHI and EagI. The samples were run on a 0.7% agarose gel, blotted onto Hybond (Amersham) nylon membrane, and hybridized to a BamHI-EagI fragment of pBR322 labeled with 32P. Quantification of the blots was performed on a PhosphorImager (Molecular Dynamics) using ImageQuant software.

Bottom Line: Eukaryotic origins of DNA replication undergo activation at various times in S-phase, allowing the genome to be duplicated in a temporally staggered fashion.Currently, there are two examples of DNA sequences that are known to advance origin activation time, centromeres and forkhead transcription factor binding sites.By combining deletion and linker scanning mutational analysis with two-dimensional gel electrophoresis to measure fork direction in the context of a two-origin plasmid, we have identified and characterized a 19- to 23-bp and a larger 584-bp DNA sequence that are capable of advancing origin activation time.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Cellular Biology Program, University of Washington, Seattle, Washington 98195.

ABSTRACT
Eukaryotic origins of DNA replication undergo activation at various times in S-phase, allowing the genome to be duplicated in a temporally staggered fashion. In the budding yeast Saccharomyces cerevisiae, the activation times of individual origins are not intrinsic to those origins but are instead governed by surrounding sequences. Currently, there are two examples of DNA sequences that are known to advance origin activation time, centromeres and forkhead transcription factor binding sites. By combining deletion and linker scanning mutational analysis with two-dimensional gel electrophoresis to measure fork direction in the context of a two-origin plasmid, we have identified and characterized a 19- to 23-bp and a larger 584-bp DNA sequence that are capable of advancing origin activation time.

Show MeSH
Related in: MedlinePlus