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

Proc Natl Acad Sci U S A. 2002 Jan 8;99(1):101-6. doi: 10.1073/pnas.012578499. Epub 2001 Dec 26.

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Binding Sites
  • Cell Cycle Proteins / chemistry
  • Cell Cycle Proteins / metabolism
  • Chromatin / metabolism
  • Chromosomes / metabolism
  • DNA / chemistry*
  • Deoxyribonuclease I / metabolism
  • Fungal Proteins / metabolism
  • Gene Library
  • Mutation
  • Nucleic Acid Conformation
  • Plasmids / metabolism
  • Precipitin Tests
  • Protein Binding
  • Replication Origin*
  • Saccharomyces cerevisiae / genetics*
  • Saccharomyces cerevisiae Proteins*

Substances

  • CDC6 protein, S cerevisiae
  • Cell Cycle Proteins
  • Chromatin
  • Fungal Proteins
  • Saccharomyces cerevisiae Proteins
  • DNA
  • Deoxyribonuclease I