Systematic mapping of autonomously replicating sequences on chromosome V ofSaccharomyces cerevisiae using a novel strategy

Abstract

We have developed a new procedure for easy and rapid identification of autonomously replicating sequences (ARSs) and have applied it to the analysis of chromosome V of Saccharomyces cerevisiae. The procedure makes use of the ordered λ phage clone bank of this chromosome that we have constructed, and includes transposition of a mini‐transposon and selection of transposon‐containing derivatives, isolation of their DNA and circularization at their cos‐ends, transformation of yeast cells with the circularized DNA, and scoring transformation frequency. The transposon used was derived from Tn5supF, contained the yeast LEU2 gene, and was placed, together with the hyperactive transposase gene, on a mini‐F plasmid for stable maintenance in Escherichia coli K‐12. Sixteen regions of chromosome V showing ARS activity were identified, of which 12 were newly found in this work. Thus, the procedure will be useful for systematic genomic scale analysis of ARSs in yeast and related organisms in which ordered clone banks have been established. The average distance between adjacent ARS‐containing regions was approximately 40 kb. Two‐dimensional gel electrophoretic analysis of chromosome replication indicated that one of the newly identified ARSs was functional as an actual in situ replication origin, at least under the conditions employed.

Chromosome integrity inSaccharomyces cerevisiae: the interplay of DNA replication initiation factors, elongation factors, and origins

“…However, selective pressures against increasing origin numbers must also exist because in budding yeast, as in many organisms, origins are spaced approximately every 40 kb (Newlon and Burke 1980;Tanaka et al 1996;Poloumienko et al 2001). Here, we show that YACs with more origins have a higher rate of GCR in orc5-70 and orc3-70 mutants.…”

Association of RPA with chromosomal replication origins requires an Mcm protein, and is regulated by Rad53, and cyclin- and Dbf4-dependent kinases

“…( A ) Genomic intervals around or at ARS501 amplified by PCR primers. The available data suggest that there are no active replication origins except ARS501 within the region of chromosome V depicted here (Ferguson et al ., 1991; Tanaka et al ., 1996). ( B ) Association of Mcm7‐myc with ARS1‐ (top), ARS305‐ (middle) and ARS501‐ (bottom) containing fragments.…”

“…A key question is whether the ARS-containing fragments that we detect in RPA immunoprecipitates in the presence of HU represents loading of RPA prior to the synthesis of DNA chains or whether it merely represents RPA present at replication forks whose movement amplified by PCR primers. The available data suggest that there are no active replication origins except ARS501 within the region of chromosome V depicted here (Ferguson et al, 1991;Tanaka et al, 1996). (B) Association of Mcm7-myc with ARS1-(top), ARS305-(middle) and ARS501-(bottom) containing fragments.…”

Early Replication of Short Telomeres in Budding Yeast

“…Analysis of replication timing in strains carrying either 0 or 16 Rap1-binding sites at the modified telomere on chr V-R revealed the expected replication pattern for the ARS elements examined (Figure 4B, top panel) with one notable difference: a shift to earlier replication timing (beginning at 40 min, instead of the usual 60 min) for ARS522 in the presence of a shortened chr V-R telomere (Figure 4B, top panel, right). Interestingly, ARS522 (also known as ARS501) is the first origin encountered inwards from the modified telomere on the well-characterized chr V (Tanaka et al, 1996). Thus, this result suggests that shortening of the telomere led to relief of the normal late replication timing imposed by telomeres on subtelomeric origins (McCarroll and Fangman, 1988).…”