Context-Dependent Modulation of Replication Activity ofSaccharomyces cerevisiaeAutonomously Replicating Sequences by Transcription Factors

Abstract

Evidence for transcription factor involvement in the initiation of DNA replication at certain replication origins in Saccharomyces cerevisiae mainly comes from an indirect assay which measures the mitotic stability of plasmids containing an autonomously replicating sequence (ARS), a selectable marker gene, and a centromere. In order to eliminate the effect of transcription factor binding to the selectable marker gene or centromere in such assays, we have adapted the DpnI assay to directly measure ARS replication activity in vivo by using ARS plasmids devoid of extraneous transcription elements. Using this assay, we found that the B3 element of ARS1, which serves as a binding site for the transcription factor Abf1p, does not stimulate ARS activity on plasmids lacking a centromere and a selectable marker gene. We also found with such plasmids that exogenous expression of the strong transcriptional activators Gal4 and Gal4-VP16 inhibited the replication activity of ARS1 when B3 was replaced by the Gal4 binding site, although these activators had previously been shown to stimulate replication activity in the stability assay. Moreover, a chromosomally inactive ARS, ARS301, which was active by itself on a plasmid, was inactivated by placing an Abf1p binding site in its vicinity. These results indicate that the sequences surrounding the ARS as well as properties of the ARS element itself determine its response to transcription factors.Saccharomyces cerevisiae provides an excellent system for the analysis of eukaryotic replication origins for several reasons. First of all, the replication origin can be identified on a functional basis by analyzing it for DNA sequences that replicate in an autonomous fashion as plasmid DNA in yeast cells (autonomous replicating sequence [ARS]). When a DNA fragment with origin activity is ligated to a selectable marker gene, the resulting plasmid can transform yeast cells with high frequency (19,43). Secondly, relative replication activity is easily estimated by measuring the mitotic stability of each ARS plasmid (24). This method has revealed that replication origins of budding yeast are compact (100 to 200 bp), with a modular structure consisting of an essential "core" sequence and auxiliary sequences which modulate replication activity. For example, ARS1, one of the best-characterized origins, contains two elements, A and B (10, 30). The A element contains a small sequence that is essential for origin activity and conserved among all ARS sequences (ARS consensus sequence [ACS]) (32). The A element functions as a recognition site for the origin recognition complex (ORC) involved in the initiation of DNA replication (3, 4). The B element consists of three subelements, B1, B2, and B3, each of which contributes to efficient replication although none is essential (30). One function of the B1 element is to enhance the binding of the ORC to the A element (34-36). The function of B2 remains unclear. The B3 element is a binding site for the transcription factor Abf1p (30). Other ARS elements so...

Transcription Factor Binding and Induced Transcription Alter Chromosomal c-myc Replicator Activity

“…Transcribed sequences are generally replicated earlier in S phase in the cells where they are expressed than in cells where they are not (22,76), possibly as a result of the modification of chromatin structure by transcription factors (48). However, several investigators report positive (11,19,21,25,34,36,47,53,66,86) or negative (11,43,53) effects of transcription factors on DNA replication, either directly through the binding of replication proteins or indirectly via modulation of chromatin structure. Alternatively, it has been suggested that cells may replicate active sequences prior to gene expression as a mechanism to avoid disruption of pre-RCs by the passage of the transcriptional machinery (12,28,57).…”

“…Transcribed sequences are generally replicated earlier in S phase in the cells where they are expressed than in cells where they are not (22,76), possibly as a result of the modification of chromatin structure by transcription factors (48). However, several investigators report positive (11,19,21,25,34,36,47,53,66,86) or negative (11,43,53) …”

Isolation and characterization of the ecdysone receptor and its heterodimeric partner ultraspiracle through development in Sciara coprophila

“…Viral transcription factors have been shown to directly recruit replication factors, such as the polyomavirus recruiting the viral large T antigen replication initiator complex with the AP1 transcription factor (Murakami et al 1991; Ito et al 1996) and the Epstein–Barr virus recruiting cellular ORC with the EBNA1 transcription factor (Chaudhuri et al 2001; Dhar et al 2001 Schepers et al 2001; Ritzi et al 2003). Many origins in Saccharomyces cerevisiae (autonomously replicating sequences, ARS) contain binding sites for transcription factors (Li et al 1998; Hu et al 1999; Kohzaki et al 1999) including the B3 element of ARS1 which enhances ARS activity and is bound by the transcription factor, Abf1 (Mahrahrens and Stillman 1992). Interestingly, both the S-phase promoting transcription factor complex, E2F-Rb (Royzman et al 1999; Bosco et al 2001; Cayirlioglu et al 2001, 2003) and the Myb transcription factor complex (Beall et al 2002; Beall et al 2004) have been implicated in regulating DNA amplification loci in Drosophila follicle cells.…”

Mcm1 Promotes Replication Initiation by Binding Specific Elements at Replication Origins

“…In mammalian cells, initiation occurs at multiple sites within replication zones that are defined by their chromosomal contexts rather than nucleotide sequences (29,46). Indeed, the activity of replication origins is responsive to their contexts (9,36). It has been shown that replication origins taken out of their native environment are no longer temporally regulated (35,52) and that silent replication origins are no longer repressed (23).…”