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.
Highlights
The DNA binding properties of ABFZ, an abundant protein, which binds to DNA as a dimer
The packaging of DNA poses a general problem: How can the accessibility of key regulatory DNA sequences within the genome to specific trans-acting factors be guaranteed against the overwhelming backdrop of nonspecific DNA binding by the packaging proteins? In this report, we show that ABFS appears to bind to DNA by wrapping and, appears capable of compacting DNA
Era1 lines of evidence argue that ABFB is an important struc- within REP2, there areonly two regions of signifitural element of the yeast mitochondrial nucleoid
Summary
Plnsmids and Strains-ABFP was purified from the yeast strains BJ 1991 (Mata leu u r d trpl prb1-1122pep4) or BJ405 (Mata trpl prbl prcl pep). The plasmid pARS1.4.1 has been previously described [11]. This plasmid contains ARSl sequences from the RsaI site at nucleotide 927 to the HinfI site a t nucleotide 734 [12] bluntended and cloned into the SmaI site of pUC19 such that domain A of ARSl [13] lies on the EcoRI side of the polylinker and domain B [13] lies on the Hind111 side of the polylinker. Of DNA replication [18] cloned into the SmaI site of pUC118 such Samples were resuspended in 10 mM Tris, pH 7.4,1 mM EDTA (TE). The plasmid pC2G1 was derived EDTA [21] buffer containing the indicated amounts of chloroquine. From C2Gl-S200, a gift from Virginia Van Houten and Carol Newlon (University of Medicine and Dentistry of NewJersey), which contains
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