Activation of a Mammalian Origin of Replication by Chromosomal Rearrangement

Abstract

The methotrexate-resistant Chinese hamster cell line DC3F/A3-4K (A3/4K) contains at least two prominent dihydrofolate reductase amplicon types. The type I amplicons, constituting ~80% of the total, are at least 650 kb in length, but the endpoints have not yet been characterized. The type II sequences represent ~20% of amplicons, are 450 kb in length, and are arranged as alternating head-to-head and tail-to-tail repeats. In previous studies on the CHOC 400 line, in which the amplicons are much smaller, a replication initiation locus (οri-β/οri-γ) has been shown to reside downstream from the dihydrofolate reductase gene. In a more recent study on the larger amplicons of A3/4K cells, we detected an additional initiation locus (ori-α) lying ~240 kb upstream from οri-β/οri-γ. Interestingly, in vivo labelling experiments suggested that replication forks diverge from ori-α only in the downstream direction. This finding suggested either that ori-cx is a unidirectional origin or that a terminus lies immediately upstream from ori-α. However, in this study, we show that ori-α is actually very close to the head-to-head palindromic junction sequence between the minor type II amplicons in A3/4K cells; furthermore, ori-α is active in the early S period in the type II amplicons but not in the larger type I sequences that lack this palindromic junction. This is the first direct demonstration in mammalian cells that a cryptic origin can be activated by chromosomal rearrangement, presumably by deleting negative regulatory elements or by creating a more favorable chromosomal milieu for initiation.