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DNA binding properties of an HMG1-related protein from yeast mitochondria
1Imperial Cancer Research Fund, Clare Hall Laboratories, Potters Bar, Hertsfordshire, United Kingdom.
Abstract:
The DNA binding properties of ABF2, an abundant protein found in the mitochondria of the yeast Saccharomyces cerevisiae have been examined in detail. ABF2 is closely related to the vertebrate high mobility group protein HMG1 and like HMG1, ABF2 will introduce negative supercoils into a relaxed, double-stranded circular DNA molecule in cooperation with a DNA topoisomerase. Additionally, ABF2 binds approximately 5-10 times more tightly to negatively supercoiled DNA than to relaxed circular or linear DNA. Although ABF2 binds to most random double-stranded sequences with roughly equal affinity, its binding within certain key regulatory regions is qualitatively quite different. First, ABF2 binding induces a distinct pattern of DNA bending within the chromosomal origin of DNA replication, ARS1. Second, ABF2 binding to all nuclear replication origins tested, in addition to a critical mitochondrial promoter and replication origin, is clearly nonrandom as visualized by DNase1 footprinting. Analysis of the sequences found within these regions as well as competition experiments with synthetic DNA molecules suggest that site-specific DNA binding may be accomplished by the phased distribution of short stretches of poly(dA), which exclude ABF2 binding. These patterns of ABF2 DNA binding suggest a role for the protein in genome organization and site-specific regulation of transcription or DNA replication.
Insights
ABF2 protein in yeast mitochondria binds DNA, influencing genome organization. It preferentially binds supercoiled DNA and specific regulatory regions, suggesting roles in replication and transcription.
Area of Science:
- Molecular Biology
- Yeast Genetics
- Biochemistry
Background:
- ABF2 is an abundant mitochondrial protein in Saccharomyces cerevisiae.
- It shares similarities with the high mobility group protein HMG1.
Purpose of the Study:
- To investigate the DNA binding properties of the yeast mitochondrial protein ABF2.
- To understand ABF2's role in DNA binding and potential regulatory functions.
Main Methods:
- DNA supercoiling assays with DNA topoisomerase.
- DNA binding affinity studies using supercoiled, relaxed circular, and linear DNA.
- DNase1 footprinting to analyze binding to regulatory regions.
- Competition experiments with synthetic DNA molecules.
Main Results:
- ABF2 introduces negative supercoils into DNA in cooperation with DNA topoisomerase.
- ABF2 exhibits higher affinity for negatively supercoiled DNA compared to relaxed or linear DNA.
- ABF2 binding induces DNA bending at the ARS1 replication origin and shows nonrandom binding at nuclear replication origins and a mitochondrial promoter/origin.
- Site-specific binding is potentially mediated by poly(dA) sequences that exclude ABF2.
Conclusions:
- ABF2 plays a role in organizing the yeast genome.
- ABF2 is involved in the site-specific regulation of transcription and DNA replication within yeast mitochondria and nucleus.