Understanding cell division requires knowing not only how, but also what determines when cells divide. Previous studies identified several components of the machinery that drives the cell cycle. However, it is not clear how cellular pathways impinge on the cell division machinery to initiate cell division. This is a critical gap in our understanding, since this process governs overall proliferation: once cells initiate their division, they are committed to completing it.
In proliferating cells, the G1 phase of any given cell cycle lasts from the end of the previous mitosis until the beginning of DNA synthesis. In unfavorable growth conditions, eukaryotic cells typically stay longer in G1, delaying initiation of DNA replication 
. Subsequent cell cycle transitions, culminating with mitosis, are less sensitive to growth limitations, and their timing does not vary greatly, even if growth conditions worsen. Hence, differences in the length of the G1 phase account for most of the differences in total cell cycle, or generation times, between the same cells growing in different media, or among different cells of the same organism. Such fundamental observations support the notion that eukaryotic cells commit to a new round of cell division at some point in late G1 
. Budding yeast cells also evaluate their “growth” in late G1 at a point called START, before DNA synthesis in S phase 
. In favorable growth conditions, and in the absence of mating pheromones (for haploids), or meiotic inducers (for diploids), cells pass through START 
. Passage through START and commitment to cell division precedes a large transcriptional program and additional events that lead to initiation of DNA replication 
The lack of a detailed view of upstream regulatory networks that govern the timing of START in the yeast Saccharomyces cerevisiae
is surprising, given the rich history of the field. The classic cdc
screen identified factors essential for START, such as Cdc28p 
, the main yeast cyclin-dependent kinase (Cdk). However, the cdc
screen did not target nonessential regulators, such as the cyclin regulatory subunits of Cdc28p 
. Other efforts relied on gene-specific suppression 
or sensitivity to mating pheromones 
. By far, however, most approaches to identify regulators of START interrogated cell size. Almost half a century ago, a relationship between the size or mass of a cell and the timing of initiation of DNA replication was described from bacterial 
, to mammalian cells 
. Indeed, a newborn budding yeast cell is smaller than its mother is, and it will not initiate cell division until it becomes bigger 
. Thus, it appears that there is a critical size threshold for START completion in yeast. Based on this concept of a critical size, the question of “when
do cells divide?” was reduced to “what size
are cells when they divide?” Hence, several screens for regulators of START interrogated cell size 
. In fact, systematic, genome-wide approaches to find genes required for the correct timing of START relied solely on cell size changes 
Any gene deletion that alters the length of the G1 phase relative to the rest of the phases of the cell cycle will alter the DNA content profile. Thus, the DNA content of a population reports on the relative length of the G1 phase directly, discerning cells with unreplicated genome. In yeast, DNA content analyses measured the effects of gene over-expression on cell cycle progression 
, or cycle arrest when essential genes were turned-off 
. However, the yeast single-gene deletion collections have not been evaluated with this method.
To assess cell cycle progression more directly, we evaluated the yeast deletion collection of nonessential genes for altered DNA content, by flow cytometry. We found that most gene deletions that altered cell cycle progression did not change cell size. Our results suggest that evaluating the length of the G1 phase of the cell cycle, instead of cell size, provides a much more accurate view of the contribution of individual gene products to the timing of START and commitment to cell division. We also documented a strong requirement for ribosomal biogenesis for initiation of cell division, and identified numerous factors that have not been implicated previously in cell cycle control mechanisms. One such factor is the metabolic enzyme cystathionine-β-synthase (CBS; Cys4p in yeast). We discovered a novel, non-catalytic role of CBS, in accelerating START.
Taken together, the data we present here substantially expand the range of factors that affect initiation of cell division. We discuss the significance of our finding that most gene deletions that change the length of the G1 phase do not alter cell size, in the context of models that center on the role of cell size at START.