Replisome loading reduces chromatin motion independent of DNA synthesis
Chromatin has been shown to undergo diffusional motion, which is affected during gene transcription by RNA polymerase activity. However, the relationship between chromatin mobility and other genomic processes remains unclear. Hence, we set out to label the DNA directly in a sequence unbiased manner and followed labeled chromatin dynamics in interphase human cells expressing GFP-tagged PCNA, a cell cycle marker and core component of the DNA replication machinery. We detected decreased chromatin mobility during the S-phase compared to G1 and G2 phases in tumor as well as normal diploid cells using automated particle tracking. To gain insight into the dynamical organization of the genome during DNA replication, we determined labeled chromatin domain sizes and analyzed their motion in replicating cells. By correlating chromatin mobility proximal to the active sites of DNA synthesis, we showed that chromatin motion was locally constrained at the sites of DNA replication. Furthermore, inhibiting DNA synthesis led to increased loading of DNA polymerases. This was accompanied by accumulation of the single-stranded DNA binding protein on the chromatin and activation of DNA helicases further restricting local chromatin motion. We, therefore, propose that it is the loading of replisomes but not their catalytic activity that reduces the dynamics of replicating chromatin segments in the S-phase as well as their accessibility and probability of interactions with other genomic regions. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/531331v2_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@ed9743org.highwire.dtl.DTLVardef@65c9d4org.highwire.dtl.DTLVardef@a583a7org.highwire.dtl.DTLVardef@6681b1_HPS_FORMAT_FIGEXP M_FIG C_FIG Highlights- Direct and sequence unbiased labeling of DNA genome-wide - DNA labeled chromatin is more mobile in G1/G2 relative to the S-phase - Restriction of chromatin motion occurs proximal to sites of DNA replication - Loading of replisomes, even in the absence of processive DNA synthesis, restricts chromatin motion