![]() We identified 796 origins that fired in cdc18 cdt1 co-oe ( supplementary material Table S2 and Table S3A). First, we mapped the origins that become activated, using the DNA synthesis inhibitor hydroxyurea (HU) to inhibit fork progression and estimating the DNA content in the vicinity of fired origins by using whole genome DNA microarrays ( Heichinger et al., 2006) ( Fig. Since this pattern of DNA synthesis did not resemble a normal S phase, we asked whether normal S phase origins of replication were being used to replicate the genome and whether they fired in a coordinated fashion. Rather, it induces an extended period of continued DNA synthesis, resulting in approximately four doublings in DNA content. S1C) ( Kiang et al., 2009) as cells increased their DNA content from 2C to 32C, we found that cdc18 cdt1 co-oe does not show characteristics of repeated, periodic S phases that require G1–S gene expression. S1A,B) ( Sivakumar et al., 2004) or observing the G1–S gene expression marker Tos4-GFP ( supplementary material Fig. We analyzed the pattern of DNA synthesis and G1–S gene expression in the presence of excess Cdc18 and Cdt1. Therefore, we asked whether genome-wide coordination of origin firing is lost in cdc18 cdt1 co-oe, leading to local amplification, and if so, what features of replication origins might be responsible for that amplification. In these cells, the S phase controls, ensuring that an origin fires no more than once per round of replication, may be abrogated, because overexpression of a cdc18 phosphorylation-site mutant brings about some local amplification, particularly at the telomeres ( Mickle et al., 2007). In fission yeast, overexpression of the CDC6 homologue cdc18 in G2 induces re-initiation of DNA synthesis up to a DNA content of about 8C–16C ( Nishitani and Nurse, 1995) during co-overexpression with cdt1 ( cdc18 cdt1 co-oe), DNA content both increases more rapidly and attains a higher ploidy level (~32C) ( Gopalakrishnan et al., 2001 Nishitani et al., 2000 Yanow et al., 2001) (see supplementary material Table S1 for genotypes). ![]() Cdc6 and Cdt1 are components of the pre-replicative complex (Pre-RC) ( Bell and Dutta, 2002 Kelly et al., 1993) that bind at replication origins and recruit the mini chromosome maintenance (MCM) complex, the likely replicative helicase ( Bell and Dutta, 2002). Second, dysregulation of the replication factors Cdc6 and Cdt1 brings about increased DNA content in a range of organisms ( Arias and Walter, 2007 Gonzalez et al., 2006 Melixetian et al., 2004 Mihaylov et al., 2002 Vaziri et al., 2003). In fission yeast Schizosaccharomyces pombe this occurs by a largely normal S phase program given that normal S phase origins are used, periodic rounds of DNA synthesis are correlated with normal G1–S gene expression and cell mass doubling, and there is even replication of the genome ( Kiang et al., 2009). First, depletion of G2–M cyclin-dependent kinase (CDK) activity in yeast and fruit fly, and mouse and human cell lines leads to repeated DNA doublings without mitosis ( Arias and Walter, 2007 Kiang et al., 2009 Mihaylov et al., 2002). Unscheduled DNA synthesis can lead to local amplification ( Arias and Walter, 2007 Varshavsky, 1981) and genome instability ( Schimke et al., 1986 Seo et al., 2005 Varshavsky, 1981), and can be induced by two types of perturbation. We propose that these features predispose replication origins to re-fire within a single S phase, or to remain active after passive replication.įor stable genome inheritance, S phase occurs once per cell cycle, and within S phase the genome is completely and evenly replicated. Origins associated with amplification are highly AT-rich, fire efficiently and early during mitotic S phase, and are located in large intergenic regions. Specific origins are necessary for amplification but act only within a permissive chromosomal context. We find that S phase controls are attenuated and coordination of origin firing is lost, resulting in local amplification. To identify features of replication origins important for such amplification, we have investigated origin firing and local genome amplification in the presence of excess helicase loaders Cdc18 and Cdt1 in fission yeast. Failure in these controls can lead to local amplification, contributing to genome instability and the development of cancer. ![]() To ensure equal replication of the genome in every eukaryotic cell cycle, replication origins fire only once each S phase and do not fire after passive replication.
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