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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

Scanning mutagenesis analysis for sequences involved in initiation bias. (A) The wild-type sequence of the SmaI-HaeIII fragment of URA3 is shown in capital letters. Sequences of the mutations are given in lowercase letters beneath the wild-type sequences they replace. The two overlined nucleotides were not changed by the mutagenesis. The boxes identify 11 of 13 bp matches to Rap1 or Abf1 consensus binding sites as well as matches to Fkh1 and Fkh2. The 3′ end of the URA3 coding sequence is indicated by the single letter amino acid code. The locations of the SmaI, NsiI, and HaeIII restriction enzyme sites are indicated. Sequences that when mutated significantly reduce the ARS1S bias are indicated in red. (B) Values for percent initiation at ARS1S are plotted for the 10 mutations that span the SmaI-HaeIII fragment. The bar on the right of the graph indicates a 99% confidence interval extending on both sides of the mean percent initiation at ARS1S for pN&S based on five separate experiments. Values that fall outside of this range are considered significantly different from pN&S at the 1% level. The approximate locations of the mutations relative to the 3′ half of the URA3 gene (arrow) are shown. (C) Fork-direction gel analysis of mutants m6 and m7 as examples of the mutations that eliminate the origin bias. (D) The wild-type sequence corresponding to the sequences of the URA3 fragment that were mutated in m5, m6, and m7 are shown in capital letters. Sequences of three different mutations (m12, ∆NsiI, m11) are given in lowercase letters beneath the wild-type sequences they replace. Deleted bases are depicted by a dash. The overlined nucleotides were not changed by the mutagenesis while the underlined nucleotides mark the NsiI site. Sequences important for the bias are indicated in red letters. Percent initiation at ARS1S is indicated as bar graphs for the three mutations, m11, m12, and ∆NsiI. (E) Fork direction 2-D gels of mutants m11 and m12 (see panel D for sequences). Percent initiation at ARS1S is indicated in the upper right corners.
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fig3: Scanning mutagenesis analysis for sequences involved in initiation bias. (A) The wild-type sequence of the SmaI-HaeIII fragment of URA3 is shown in capital letters. Sequences of the mutations are given in lowercase letters beneath the wild-type sequences they replace. The two overlined nucleotides were not changed by the mutagenesis. The boxes identify 11 of 13 bp matches to Rap1 or Abf1 consensus binding sites as well as matches to Fkh1 and Fkh2. The 3′ end of the URA3 coding sequence is indicated by the single letter amino acid code. The locations of the SmaI, NsiI, and HaeIII restriction enzyme sites are indicated. Sequences that when mutated significantly reduce the ARS1S bias are indicated in red. (B) Values for percent initiation at ARS1S are plotted for the 10 mutations that span the SmaI-HaeIII fragment. The bar on the right of the graph indicates a 99% confidence interval extending on both sides of the mean percent initiation at ARS1S for pN&S based on five separate experiments. Values that fall outside of this range are considered significantly different from pN&S at the 1% level. The approximate locations of the mutations relative to the 3′ half of the URA3 gene (arrow) are shown. (C) Fork-direction gel analysis of mutants m6 and m7 as examples of the mutations that eliminate the origin bias. (D) The wild-type sequence corresponding to the sequences of the URA3 fragment that were mutated in m5, m6, and m7 are shown in capital letters. Sequences of three different mutations (m12, ∆NsiI, m11) are given in lowercase letters beneath the wild-type sequences they replace. Deleted bases are depicted by a dash. The overlined nucleotides were not changed by the mutagenesis while the underlined nucleotides mark the NsiI site. Sequences important for the bias are indicated in red letters. Percent initiation at ARS1S is indicated as bar graphs for the three mutations, m11, m12, and ∆NsiI. (E) Fork direction 2-D gels of mutants m11 and m12 (see panel D for sequences). Percent initiation at ARS1S is indicated in the upper right corners.

Mentions: The revertible selectable marker method was used for mutagenesis (Deng and Nickoloff 1992). Oligonucleotides were purchased from Gibco BRL. The sequences of the mutations m1-10 are given in Figure 3A. The sequences of the mutations m11, m12, and ∆NsiI are given in Figure 3D. The sequences of the DNA unwinding elements (DUE) mutations corresponding to the m5-m7 positions were m5DUE (ATCAAGTACT; data not shown), m6DUE (ATAGACGTCA; Supporting Information, Figure S2), and m7DUE (ATTAATAGT; Figure S2). Correct transformants were identified by restriction digestion screening for incorporation of a restriction site (XhoI for m1-10; ScaI for m5DUE; AatII for M6DUE; AseI for m7DUE), and the mutations were confirmed by sequencing. The success rate of the mutagenesis was generally approximately 60%.


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)

Scanning mutagenesis analysis for sequences involved in initiation bias. (A) The wild-type sequence of the SmaI-HaeIII fragment of URA3 is shown in capital letters. Sequences of the mutations are given in lowercase letters beneath the wild-type sequences they replace. The two overlined nucleotides were not changed by the mutagenesis. The boxes identify 11 of 13 bp matches to Rap1 or Abf1 consensus binding sites as well as matches to Fkh1 and Fkh2. The 3′ end of the URA3 coding sequence is indicated by the single letter amino acid code. The locations of the SmaI, NsiI, and HaeIII restriction enzyme sites are indicated. Sequences that when mutated significantly reduce the ARS1S bias are indicated in red. (B) Values for percent initiation at ARS1S are plotted for the 10 mutations that span the SmaI-HaeIII fragment. The bar on the right of the graph indicates a 99% confidence interval extending on both sides of the mean percent initiation at ARS1S for pN&S based on five separate experiments. Values that fall outside of this range are considered significantly different from pN&S at the 1% level. The approximate locations of the mutations relative to the 3′ half of the URA3 gene (arrow) are shown. (C) Fork-direction gel analysis of mutants m6 and m7 as examples of the mutations that eliminate the origin bias. (D) The wild-type sequence corresponding to the sequences of the URA3 fragment that were mutated in m5, m6, and m7 are shown in capital letters. Sequences of three different mutations (m12, ∆NsiI, m11) are given in lowercase letters beneath the wild-type sequences they replace. Deleted bases are depicted by a dash. The overlined nucleotides were not changed by the mutagenesis while the underlined nucleotides mark the NsiI site. Sequences important for the bias are indicated in red letters. Percent initiation at ARS1S is indicated as bar graphs for the three mutations, m11, m12, and ∆NsiI. (E) Fork direction 2-D gels of mutants m11 and m12 (see panel D for sequences). Percent initiation at ARS1S is indicated in the upper right corners.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3815058&req=5

fig3: Scanning mutagenesis analysis for sequences involved in initiation bias. (A) The wild-type sequence of the SmaI-HaeIII fragment of URA3 is shown in capital letters. Sequences of the mutations are given in lowercase letters beneath the wild-type sequences they replace. The two overlined nucleotides were not changed by the mutagenesis. The boxes identify 11 of 13 bp matches to Rap1 or Abf1 consensus binding sites as well as matches to Fkh1 and Fkh2. The 3′ end of the URA3 coding sequence is indicated by the single letter amino acid code. The locations of the SmaI, NsiI, and HaeIII restriction enzyme sites are indicated. Sequences that when mutated significantly reduce the ARS1S bias are indicated in red. (B) Values for percent initiation at ARS1S are plotted for the 10 mutations that span the SmaI-HaeIII fragment. The bar on the right of the graph indicates a 99% confidence interval extending on both sides of the mean percent initiation at ARS1S for pN&S based on five separate experiments. Values that fall outside of this range are considered significantly different from pN&S at the 1% level. The approximate locations of the mutations relative to the 3′ half of the URA3 gene (arrow) are shown. (C) Fork-direction gel analysis of mutants m6 and m7 as examples of the mutations that eliminate the origin bias. (D) The wild-type sequence corresponding to the sequences of the URA3 fragment that were mutated in m5, m6, and m7 are shown in capital letters. Sequences of three different mutations (m12, ∆NsiI, m11) are given in lowercase letters beneath the wild-type sequences they replace. Deleted bases are depicted by a dash. The overlined nucleotides were not changed by the mutagenesis while the underlined nucleotides mark the NsiI site. Sequences important for the bias are indicated in red letters. Percent initiation at ARS1S is indicated as bar graphs for the three mutations, m11, m12, and ∆NsiI. (E) Fork direction 2-D gels of mutants m11 and m12 (see panel D for sequences). Percent initiation at ARS1S is indicated in the upper right corners.
Mentions: The revertible selectable marker method was used for mutagenesis (Deng and Nickoloff 1992). Oligonucleotides were purchased from Gibco BRL. The sequences of the mutations m1-10 are given in Figure 3A. The sequences of the mutations m11, m12, and ∆NsiI are given in Figure 3D. The sequences of the DNA unwinding elements (DUE) mutations corresponding to the m5-m7 positions were m5DUE (ATCAAGTACT; data not shown), m6DUE (ATAGACGTCA; Supporting Information, Figure S2), and m7DUE (ATTAATAGT; Figure S2). Correct transformants were identified by restriction digestion screening for incorporation of a restriction site (XhoI for m1-10; ScaI for m5DUE; AatII for M6DUE; AseI for m7DUE), and the mutations were confirmed by sequencing. The success rate of the mutagenesis was generally approximately 60%.

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