DNA Replication Origin Interference Increases the Spacing between Initiation Events in Human Cells

Abstract

Mammalian DNA replication origins localize to sites that range from base pairs to tens of kilobases. A regular distribution of initiations in individual cell cycles suggests that only a limited number of these numerous potential start sites are converted into activated origins. Origin interference can silence redundant origins; however, it is currently unknown whether interference participates in spacing functional human initiation events. By using a novel hybridization strategy, genomic Morse code, on single combed DNA molecules from primary keratinocytes, we report the initiation sites present on 1.5 Mb of human chromosome 14q11.2. We confirm that initiation zones are widespread in human cells, map to intergenic regions, and contain sequence motifs found at other mammalian initiation zones. Origins used per cell cycle are less abundant than the potential sites of initiation, and their limited use increases the spacing between initiation events. Between-zone interference decreases in proportion to the distance from the active origin, whereas within-zone interference is 100% efficient. These results identify a hierarchical organization of origin activity in human cells. Functional origins govern the probability that nearby origins will fire in the context of multiple potential start sites of DNA replication, and this is mediated by origin interference. INTRODUCTIONEukaryotic cells have a limited amount of time, defined by the length of S phase, to duplicate their genomes. This is achieved by synthesizing DNA at replication forks, which extend from multiple sites of initiation. Because fork speed is not scaled according to S-phase length, regulating the frequency of initiation along each respective chromosome is required to prevent unreplicated gaps before the onset of mitosis (Hand and Tamm, 1973;Edenberg and Huberman, 1975).Although there are exceptions, the common view is that somatic mammalian origins fire at 50-to 300-kilobase (kb) intervals (Edenberg and Huberman, 1975;Berezney et al., 2000). This suggests that Metazoa do possess a mechanism to evenly distribute initiation events. Placing strong replicator sequences at regular distances is one such mechanism that is used by the budding yeast, Saccharomyces cerevisiae (Newlon et al., 1991;Shirahige et al., 1993). In higher eukaryotes, genetic elements play a role in origin activation; however, they are not sufficient by themselves to drive initiation (Gilbert, 2004). Furthermore, although some Metazoan origins localize to well-circumscribed sites of a few base pairs, a large number localize to more disperse initiation zones ranging up to tens of kbs (DePamphilis, 1999).This raises the problem of how to achieve a regular distribution of activated origins from a range of potential sites that possess low intrinsic efficiency.One method to regulate origin activity is to change the probability that it will be replicated passively. As an elongating fork from an origin neighbor mediates this suppression, this form of origin deactivation has been termed "origin inte...

Genome-Wide Analysis of the Arabidopsis Replication Timing Program

“…The sizes of segment identified in this study (Fig. 3D) are 533 comparable to the putative replicons described for Arabidopsis chromosome 4 (Lee et al, 2010) 534 and some animal systems (MacAlpine et al, 2004;Lebofsky et al, 2006;Schwaiger et al, 2009). 535…”

Defining replication origin efficiency using DNA fiber assays

“…Scale bar 100 kb b colleagues revealed that replication origins form clusters, which are distributed every 40 kb on average and are reminiscent of the "initiation zones" detected earlier with biochemical approaches (DePamphilis 1999). Only one third of these clusters fire every cell cycle, presumably because of origin interference within and between initiation zones (Lebofsky et al 2006). A similar analysis of DNA replication at the human rDNA array revealed a complex pattern of initiation events and replication pause sites (Lebofsky and Bensimon 2005).…”

“…DNA fibers can also be stretched by capillarity in microchannels generated with nanofabrication processes (Sidorova et al 2008). All these approaches can be coupled to fluorescence in situ hybridization (FISH) to identify regions of interest and monitor DNA replication at specific loci (Lebofsky et al 2006;Norio and Schildkraut 2001;Pasero et al 2002).…”

“…In this approach, an asynchronous population of cells is pulsed-labeled for a short period of time with halogenated nucleotides to determine the position of replication origins (arrowheads). DNA fibers can be counterstained with anti-ssDNA antibodies (blue) (Versini et al 2003), and specific regions can be analyzed by FISH (Lebofsky et al 2006); c DNA combing analysis of densely spaced origins in Xenopus egg extracts (Marheineke and Hyrien 2001). Initiation sites are labeled with DIG-dUTP (red) to mark replication origins (arrowheads) after release from an aphidicolin block and biotin-dUTP (green) is added at variable times until completion of DNA replication; d DNA combing analysis of interorigin distances (IODs) in yeast.…”

Overreplication of short DNA regions during S phase in human cells

“…In all these cases, and in another 28 human ORIs recently identified (Lucas et al 2007), replication origins have been found to map to discrete genomic regions. In contrast, large regions containing multiple initiation sites have also been described downstream from the hamster DHFR gene (Hamlin and Dijkwel 1995), in the mouse immunoglobulin heavy chain locus (Norio et al 2005), and in human primary keratinocytes (Lebofsky et al 2006). We analyzed in detail the initiation of DNA replication at several human ORIs associated with promoters by a new quantitative PCR (Q-PCR) approach, and we report here that ORI firing during S phase is accompanied by the overproduction of short double-stranded fragments encompassing these regions.…”