Multiple determinants controlling activation of yeast replication origins late in S phase.

Abstract

Analysis of a 131-kb segment of the left arm of yeast chromosome XIV beginning 157 kb from the telomere reveals four highly active origins of replication that initiate replication late in S phase. Previous work has shown that telomeres act as determinants for late origin activation. However, at least two of the chromosome XIV origins maintain their late activation time when located on large circular plasmids, indicating that late replication is independent of telomeres. Analysis of the replication time of plasmid derivatives containing varying amounts of chromosome XIV DNA show that a minimum of three chromosomal elements, distinct from each tested origin, contribute to late activation time. These late determinants are functionally equivalent, because duplication of one set of contributing sequences can compensate for the removal of another set. Furthermore, insertion of an origin that is normally early activated into this domain results in a shift to late activation, suggesting that the chromosome XIV origins are not unique in their ability to respond to the late determinants.[Key Words: Chromosomal domains; chromosome walk; 2D agarose gels; Saccharomyces cerevisiae] Received January 17, 1996; revised version accepted May 28, 1996.The process of DNA replication is intricately controlled and integrated within the eukaryotic cell cycle. For ex ample, the onset of DNA replication is delayed in re sponse to damage of the DNA template (Siede et al. 1993), mitosis does not ensue until replication has been completed (Weinert andHartwell 1988, 1989), and rep lication of the genome is restricted to a single round during each S phase (Blow and Laskey 1988). Additional controls are exerted over the time at which chromosom al regions initiate replication. This temporal pattern can be visualized in mammalian cells by incorporating BrdU into DNA at discrete times during S phase and examin ing the chromosomes at mitois (e.g., Drouin et al. 1990). BrdU incorporation reveals interspersed early and latereplicating regions. The average DNA content of these regions is estimated at 2500-3000 kb, an amount suffi cient to contain 10-50 origins of replication (Drouin et al. 1990). Because most neighboring origins have been found by DNA fiber autoradiography to initiate replica tion fairly synchronously (Hand 1975), the origins loThese authors contributed equally to this work.

Conservation of replication timing reveals global and local regulation of replication origin activity

“…These origins offer insight into the mechanisms that regulate origin activity, such as the time during S phase when the origin activates. Long-range global mechanisms that are involved in the regulation of origin activity include chromatin modifications (Friedman et al 1996;Vogelauer et al 2002;Aparicio et al 2004) and proximity to a telomere or a centromere (Raghuraman et al 2001;Pohl et al 2012). Consistent with these findings, we observe that shorter telomeres in a S. cerevisiae/S.…”

Replication profile of Saccharomyces cerevisiae chromosome VI

“…Furthermore, the few origins randomly identified on other chromosomes had generally been found to be very efficient (e.g. Ferguson et al 1991;Friedman et al 1996), suggesting that most origins were used in nearly all cell cycles. In contrast, the survey of chromosome VI origins suggests that, at least for a small chromosome, extremely efficient origins are in the minority.…”

“…In order to normalize between experiments, the T rep is determined for early and late replicating reference fragments. The Replication Index (RI; Friedman et al 1996) of the ARS603.5 KL Friedman et al 672 Genes to Cells (1997) 2, 667-678 was not determined because this origin is not active (see Fig. 1).…”

“…Similarly, we have recently identified four origins on chromosome XIV that are activated late in S phase. These origins are located more than 170 kb from the nearest telomere, and remain late-replicating when located on a circular plasmid (Friedman et al 1996). Sequences surrounding these late origins influence activation time, although the mechanism by which these sequences affect origin activation time is not understood.…”

Chromatin remodelling and DNA replication: from nucleosomes to loop domains

“…DNA is assembled into di erent chromatin structures, heterochromatin and euchromatin, which cover large areas of the genome. Heterochromatin domains are transcriptionally silent, and are replicated later than transcriptionally active euchromatin domains (Deuring et al, 2000;Friedman et al, 1996;Kornberg and Lorch, 1995;Leach et al, 2000). The basic units of chromatin, nucleosomes, are separated by linker DNA that may vary in length and provides¯exibility to the nucleosome chain.…”