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DNA helical stability accounts for mutational defects in a yeast replication origin
D A Natale1, A E Schubert, D Kowalski
1Molecular and Cellular Biology Department, Roswell Park Cancer Institute, Buffalo, NY 14263.
Summary
A DNA unwinding element (DUE) is crucial for replication origin function. This study identifies a DUE in the C2G1 ARS, demonstrating its role in DNA helical instability and replication efficiency.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Autonomously replicating sequences (ARS) are essential for DNA replication initiation.
- DNA unwinding elements (DUEs) facilitate origin unwinding and have been identified in some ARS.
- The C2G1 ARS is a chromosomal replication origin with an incompletely characterized DUE.
Purpose of the Study:
- To identify and characterize the DNA unwinding element (DUE) in the C2G1 ARS.
- To investigate the role of DNA helical stability in C2G1 ARS function.
- To determine the relationship between helical instability and replication efficiency.
Main Methods:
- Computational analysis of DNA helical stability from base sequence.
- Nuclease hypersensitivity assays to map DUE location.
- Site-directed mutagenesis to alter DNA helical stability.
- Plasmid replication assays to measure ARS activity.
Main Results:
- A computer program accurately predicted DUE location and nuclease-hypersensitive sites in the C2G1 ARS.
- The DUE spans a 100-base-pair region in the 3'-flanking area of the C2G1 ARS.
- Stabilizing mutations in the DUE region reduced or abolished ARS-mediated replication.
- Replication efficiency correlated quantitatively with the degree of helical instability.
Conclusions:
- A DNA unwinding element (DUE) is a conserved and essential component of the C2G1 ARS.
- DNA helical instability within the DUE is critical for replication origin activity.
- The DUE is a major determinant of C2G1 ARS replication efficiency.