The two faces of higher eukaryotic DNA replication origins

Abstract

Maarten H. K. Linskens and Joel A. Huberman Department of Molecular and Cellular Biology Roswell Park Cancer Institute Buffalo, New York 14263 Earlier this year, Vaughn et al. (1990) described convinc- ing evidence for the surprising conclusion that the DNA replication origin located downstream of the dihydrofolate reductase (DHFR) gene in Chinese hamster cells consists of a broad initiation zone extending over 26 kb. However, the data presented by Burhans et al. (1990) in this issue suggest the contradictory conclusion that replication forks must emanate bidirectionally from a site that is no larger than 450 nucleotides. In this review, we hope to show that these apparently conflicting data are not necessarily ir- reconcilable. Instead, the two sets of data may illuminate two different faces of the higher eukaryotic chromosomal replication origin. The existence of replication origins in mammalian cells was first suggested by DNA fiber autoradiography, which revealed that replication forks move bidirectionally out- ward from sites (origins) irregularly spaced along chro- mosomal DNA molecules. However, the resolution of fiber autoradiography (15-30 kb) is insufficient to provide infor- mation about events during initiation of replication or to determine whether origins correspond to specific nucleo- tide sequences. The subsequent mapping of replication origins in E. coli and prokaryotic and eukaryotic viruses (most notably SV40) to a few hundred nucleotides or less suggested that eukaryotic chromosomal origins might correspond to short stretches of defined nucleotide se- quence. Indeed, chromosomal replication origins in the yeast S. cerevisiae correspond to specific DNA segments of a few hundred nucleotides or less (Linskens and Huber- man, 1966). The identification of replication origins in higher eukary- otes, however, has proven to be more difficult than in yeast (reviewed in Umek et al., 1969). The complexity of the higher eukaryotic genome has been a major obstacle. To overcome this difficulty, Hamlin’ s laboratory developed a strain of Chinese hamster ovary cells containing 500- 1000 copies of 240 kb surrounding the DHFR gene. By de- termining which restriction fragments of the amplified do- main are replicated earliest in S phase, Heintz and Hamlin (1982) identified a region downstream of the DHFR gene that appeared to contain a replication origin. This region is still the only stretch of mammalian chromosomal DNA has been proven to contain a replication origin. High resolution, early S phase labeling (Burhans et al., 1986; Leu and Hamlin, 1989) and replication polarity anal- ysis (Handeli et al., 1989) have suggested the existence of two separate origins about 22 kb apart within the DHFR downstream region. Figure 1 summarizes these data. Bur- hans et al. (1990) measured Okazaki fragment polarity by an independent technique to map the left-hand origin (Figure 1) to even higher resolution. Their data suggest