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. 2006 Dec;17(12):5337-45.
doi: 10.1091/mbc.e06-04-0298. Epub 2006 Sep 27.

DNA replication origin interference increases the spacing between initiation events in human cells

Ronald Lebofsky  1 , Roland Heilig, Max Sonnleitner, Jean Weissenbach, Aaron Bensimon
Affiliations

DNA replication origin interference increases the spacing between initiation events in human cells

Ronald Lebofsky et al. Mol Biol Cell. 2006 Dec.

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.

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Figures

Figure 1.
Figure 1.
DNA replication initiation mapping on 1.5 Mb in human chromosome 14q11.2. (A) Three types of replication signals on combed DNA that indicate an origin. Initiation occurs before the labeling periods, during the IdU pulse (blue) and during the CldU pulse (red) giving rise to the signals shown in i, ii, and iii, respectively. In all three cases, the midpoint of the tracks is assumed to be the site of initiation. (B) Hybridization strategies on combed DNA. Two probes of equal length but detected with different colors (i) or two probes of different length detected with the same color (ii) are hybridized to visualize a genomic region of interest. Alternatively, gaps between probe sets can be used to provide the same information. In iii, four short probes are hybridized giving rise to three informative gaps. Gap 3 allows the molecule to be oriented during breakage (iv and v). DNA breaks are denoted by a pair of vertical solid lines. (C) GMC covering 1.356 Mb in human chromosome 14q11.2. The linear patterns of the first four symbols in Morse code, a, b, c, and d, are provided. GMC comprises these four symbols, each symbol represented by a collection of probes. Probes are shown in green. Coding gaps are short gap and long gap. Start gap and end gap were included to help orient symbols when DNA breaks. Symbols are separated by space gaps. Probe and gap sizes in kb are given above each, respectively. (D) Examples of observed initiation events in the GMC region. White arrows indicate the initiation site. For fibers 2, 7, 8, and 13, GMC is still decidable, even though all probes pertaining to a symbol are not present. Initiation events flanking the symbols were mapped when one of the adjacent symbols was decoded (molecules 1, 5, 11, and 14) or space gap information was available (molecules 4 and 10). Bar, 100 kb.
Figure 2.
Figure 2.
Initiation zone identification by cluster analysis. (A) Defining ideal clusters. Equations for within variance (W) and between variance (B) are shown in the inset. For W, N is the number of clusters and Vi is the variance of cluster i. For B, Ci is the centroid of cluster i and C̄ is the mean of all the centroids in a cluster set. Plotting B – W as a function of the number of clusters revealed maximal values when the data were divided into 9 (blue circle), 22 (green circle), and 45 (red circle) groups. (B) The breakdown of one cluster into its components when 9 (blue line), 22 (green line), and 45 (red line) partitions are applied to the data. Regions underneath the red lines represent initiation zones, which are illustrated by the white boxes above the molecules. White arrows indicate the initiation site. Bar, 100 kb. (C) Distribution of initiation zones in the GMC region. The horizontal red lines denote zone size and position. Vertical white lines designate positions of all the 307 initiation sites mapped. Zones that contain fewer than four data points are marked by a horizontal gray line instead of a red line and are not considered in subsequent analyses. A horizontal black line indicates the cluster in B. Bar, 100 kb.
Figure 3.
Figure 3.
Initiation zone correlation with gene location and sequence. Horizontal red lines denote initiation zones. Initiation events that contributed to initiation zone mapping are shown as vertical white lines. Blue rectangles illustrate gene position and size. The gene name is given above the corresponding blue rectangle. Initiation zones that lie entirely in intergenic regions look white. Light gray, dark gray, and black initiation zones represent zones that reside in 90, 50–90, and <50% intergenic regions, respectively. Sequence elements underlying the initiation zones were analyzed as follows: AT, 500-base pair elements with AT content >70%; AG, at least 50 base pairs with AG content greater than 98%; (TTA)4-5; A3-4, five A3-4 stretches each separated by 10 base pairs. The numbers of these elements found within an initiation zone are provided below each initiation zone. Bracketed AT numbers indicate AT-rich 500-bp elements with >65% AT content. Bar, 100 kb.
Figure 4.
Figure 4.
Spatiotemporal analysis of functional origins. (A) Replication signals that provide interorigin distances (X). In i, the replication tracks from two initiation sites remain separate. In ii and iii, oncoming forks merge during the IdU and CldU pulses, respectively. (B) Histogram showing the frequency of measured interorigin distances. (C) Examples of molecules with at least two initiation events in a and b (i) and c and d (ii). White arrows indicate the initiation site. Initiation zones are marked by horizontal red lines. For individual molecules, the initiation zone from which an origin fires is indicated by a white box. Dark boxes designate silent initiation zones. Bar, 100 kb.
Figure 5.
Figure 5.
Origin interference based on fork extension. (A) Forks elongating from the active origin in zone (v) cover the region bounded by the vertical line pairs. The termination of the leftward moving fork is observed (inverted open triangles). Initiation zones i and ii are not interfered with, because the fork from origin (v) does not extend to its boundaries. Zones iii, iv, and vi are suppressed as the fork passively replicates their entire lengths. The rightward moving fork penetrates zone (vii), but it does not reach its centroid (black dot). This zone is not included in the origin interference data. Gray rectangles designate initiation zones and dark gray rectangle designate suppressed initiation zones. (B) Examples of molecules that display origin interference. The initiation zones relevant to this figure are illustrated by the horizontal red lines. The white box marks the initiation zone from which origins fire (white arrows). Dark boxes indicate initiation zones that are suppressed because of fork extension. Bar, 100 kb. (C) Histogram showing the frequency of distances between an initiation event and zones interfered with. Zones that were suppressed by centromeric and telomeric moving forks are represented by negative and positive values, respectively.
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