Replication of the <i>Igh</i> locus in <i>Pax5</i>⁻^/−<i>Rag2</i>⁻^/− uncommitted pro-B cells (129/Sv).

<p>The panels containing experimental data (A–D) appear discontinuous because independent experiments were performed for each restriction fragment. (A) The top portion of this panel shows the schematics of the four restriction fragments depicted in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001360#pbio-1001360-g001" target="_blank">Figure 1A</a> (shown to scale). Red and green bars indicate the positions of the initiation and termination events detected by SMARD. Numerals indicate the normalized frequency of the events scored in each population of restriction fragments (this value represents an underestimate of the actual number of events taking place during every replication cycle, a fact that is taken in consideration by our mathematical model; see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001360#s4" target="_blank">Materials and Methods</a>). These events are organized from top to bottom by increasing size of the labeled region. Some bars are shown adjacent to each other to emphasize that they occupy non-overlapping locations; this does not mean that the events occurred on the same DNA molecule. The midpoint on each bar is marked by black symbols to indicate the most likely position of an active origin or fork collision. Gray arrows show the predominant directions of replication fork movement across the region. (B, C) Distribution of the replication forks moving leftward and rightward, from SMARD (empty symbols), and from the fit (line). Each value indicates the number of replication forks per kb (fork density) detected in the population of double-labeled DNA molecules after averaging the result over intervals of defined size (10 kb for the <i>Pme</i>I fragments, 5 kb for <i>Pac</i>I#3 and <i>Swa</i>I#2). Trends in fork direction can be detected by comparing, for individual fragments, the densities shown in (B) and (C). The densities of different fragments cannot be compared directly because this value is a function of the replication time, which varies for each fragment (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001360#pbio.1001360.s008" target="_blank">Table S1</a>, Column i). Error bars were calculated as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001360#s4" target="_blank">Materials and Methods</a>. (D) IdU content of the population of double-labeled DNA molecules averaged over intervals of defined size (see panel B), obtained experimentally by SMARD (empty symbols), and from the fit (line). For each of the four restriction fragments, individual data points indicate the fraction of double-labeled molecules substituted with IdU within that interval. Since each fragment was studied individually, discontinuities are visible. However, adjacent fragments show similar trends in the shape of their graphs (increasing or decreasing), allowing us to deduce the position of the regions replicating first (peaks) and last (valleys). Error bars were calculated as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001360#s4" target="_blank">Materials and Methods</a>. (E) Distribution of the firing rate of origins across the <i>Igh</i> locus (blue line) and at adjacent genomic locations (red and blue arrows). Changes in the firing rate are shown continuously along the 1.4 megabase region spanned by the four restriction fragments. The firing rate is expressed in initiations/kb/minute to indicate that during each minute of the S phase, within a specific 1 kb section of the locus, a certain number of origins will fire in a population of 10⁻⁶ unreplicated DNA molecules (see also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001360#pbio-1001360-t001" target="_blank">Table 1</a>). This figure also shows the efficiency of origin firing within specific sections of the <i>Igh</i> locus (gray dashed lines), with gray numerals indicating the number of initiation events occurring in each region, per allele, per S phase (IAS). (F) Curves showing extent of DNA replication at different times in S phase for the various parts of the <i>Igh</i> locus (10%, 50%, and 90% replicated).