Single-stranded-DNA-binding protein-dependent DNA unwinding of the yeast ARS1 region.

Abstract

DNA unwinding of autonomously replicating sequence 1 (ARSI) from the yeast Saccharomyces cerevisuie was investigated. When a negatively supercoiled plasmid DNA containing ARS) was digested with single-strandspecific mung bean nuclease, a discrete region in the vector DNA was preferentially digested. The origin of DNA replication is defined as the site where replication initiates. In bacteria such as Escherichia coli and Bacillus subtilis, replication initiates from a single chromosomal origin (for a review, see reference 5). In the yeast Saccharomyces cerevisiae, autonomously replicating sequences (ARS) are well-defined chromosomal origins that function in plasmids (40,45,47). ARS elements confer upon their plasmid the ability to replicate in yeast cells, allowing the plasmid to transform the yeast cells at a high frequency. A sequence comparison among identified ARS elements revealed that the ARS core consensus sequence consists of 11 bp of 5'-(T/ A)TrrA(C/T)(G/A)TT1'(T/A)-3', which is essential for origin function (8,52). Point mutations in the core consensus sequence cause the loss of ARS activity (10, 24, 52). A flanking region located adjacent to the 3' side of the T-rich strand of the consensus sequence is also required for efficient replication. The 3'-flanking region is A+T rich and contains several sequences that almost match the core consensus (41). Deletions in the flanking region lead to increases in the rate of plasmid loss, whereas point mutations in the similar core consensus sequences have little effect onARS function (24, 41), suggesting that a feature(s) other than the sequence is important for the activity.ARS1 is derived from a replication origin of chromosome IV (6). Analysis of the transformation activity of deletion mutants with mutations in ARS1 has shown that ARS1 consists of domains A, B. and C (Fig. 1) (10, 44). The A domain is a core consensus sequence that is fully matched. The 3'-flanking B domain of about 100 bp contains three 9-of-11 matched consensus sequences and a binding site for protein ABF1 (13 (32) have shown, by using linker substitution mutants, that three distinct regions (B1, B2, and B3) in the B domain are required for ARS function. The C domain of about 80 bp is unique in ARS1 and is located 200 bp away from the A domain on the opposite side from the B domain. When the C domain is deleted, the rate of plasmid loss increases two-to threefold (27,40,46). The initial step of DNA replication is the DNA unwinding at or near the origin, which provides an entry site for replication enzymes. Initiator proteins, which have been identified in bacteria and eukaryotic virus systems, facilitate the unwinding near the origin by binding to specific sequences (4, 5). Kowalski and coworkers (29,50,51) showed the DNA-unwinding property of S. cerevisiae and E. coli replication origins by using single-strand-specific nucleases as probes and proposed that the ease of DNA unwinding is one of the determinants of DNA replication origins. They showed that the 3'-flanking region of the ARS co...

DNA replication from initiation zones of mammalian cells in a model system.

“…The DNA was incubated with or without HSSB prior to the digestion, and the digested sites were determined by treating the DNA with a single-cut restriction enzyme, KpnI or SmaI, and calculating the distance from each of the restriction sites. In the absence of HSSB, the terminator region of ampicillin resistance gene was mainly digested (29) and only slight cleavage was detected in the center of c-myc initiation zone. When the DNA was incubated with HSSB, the signal from the ampicillin terminator region decreased and digestion at several other sites including the center of the initiation zone was enhanced.…”

“…merase a-primase (22,29). The requirement for T antigen is different between the two systems of c-myc origin and ARS1, in that T antigen is essential for replication from the ARS1 region.…”

“…The conditions under which DNA replication of plasmid DNA containing ARS1 was measured (21) were used for replication of HMYC and pX24 DNA (9 ,ul) at 37°C for 12 min, and then mung bean nuclease (1 RI; 50 U) was added to the mixture and then incubated for 60 min (29). After purified DNA was digested with a restriction enzyme, the 5' ends of the DNA were labeled with T4 polynucleotide kinase and°9 [-y-32P]ATP.…”

“…In this system, 6490 ISHIMI ET AL. both SV40 T antigen as a DNA helicase and HSSB interacted with the origin region where the DNA is unwound with the torsional stress of negative supercoils to initiate DNA replication in the presence of DNA polymerase ot-primase (22,29). DNA gyrase is required for the elongation step, as a DNA swivelase that relaxes positive supercoils accumulated ahead of the replication forks.…”

Trinucleotide Repeat Instability is Modulated by DNA Base Lesions and DNA Base Excision Repair

“…Thus, PARP1 plays a critical role in determining the proper BER pathway for repair. Likewise, proliferating cell nuclear antigen (PCNA) has been shown to be involved in facilitating the steps of LP-BER by stimulating the replicative polymerases and FEN1 (45,(72)(73)(74). Proliferating cell nuclear antigen may also facilitate cooperation between repair enzymes as evidenced by the direct interaction of PCNA with all of the core BER proteins, including FEN1, pol β, and LIG I (46,(73)(74)(75).…”