Bulletin of the American Physical Society
APS March Meeting 2011
Volume 56, Number 1
Monday–Friday, March 21–25, 2011; Dallas, Texas
Session X7: Quantitative Approaches to DNA Replication |
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Sponsoring Units: DBP Chair: John Bechhoefer, Simon Fraser University Room: Ballroom C3 |
Thursday, March 24, 2011 2:30PM - 3:06PM |
X7.00001: Replication domains are self-interacting structural chromatin units of human chromosomes Invited Speaker: In higher eukaryotes, the absence of specific sequence motifs marking the origins of replication has been a serious hindrance to the understanding of the mechanisms that regulate the initiation and the maintenance of the replication program in different cell types. In silico analysis of nucleotide compositional skew has predicted the existence, in the germline, of replication N-domains bordered by putative replication origins and where the skew decreases rather linearly as the signature of a progressive inversion of the average fork polarity. Here, from the demonstration that the average fork polarity can be directly extracted from the derivative of replication timing profiles, we develop a wavelet-based pattern recognition methodology to delineate replication U-domains where the replication timing profile is shaped as a U and its derivative as a N. Replication U-domains are robustly found in seven cell lines as covering a significant portion (40-50{\%}) of the human genome where the replication timing data actually displays some plasticity between cell lines. The early replication initiation zones at U-domains borders are found to be hypersensitive to DNase I cleavage, to be associated with transcriptional activity and to present a significant enrichment in insular-binding proteins CTCF, the hallmark of an open chromatin structure. A comparative analysis of genome-wide chromatin interaction (HiC) data shows that replication-U domains correspond to self-interacting structural high order chromatin units of megabase characteristic size. Taken together, these findings provide evidence that the epigenetic compartmentalization of the human genome into autonomous replication U-domains comes along with an extensive remodelling of the threedimensional chromosome architecture during development or in specific diseases. The observed cell specific conservation of the replication timing between the human and mouse genomes strongly suggests that this chromosome organization into self-interacting structural and functional units is a general feature of mammalian organisms. [Preview Abstract] |
Thursday, March 24, 2011 3:06PM - 3:42PM |
X7.00002: Thermal Replication Trap Invited Speaker: The hallmark of living matter is the replication of genetic molecules and their active storage against diffusion. We have argued in the past that thermal convection can host the million-fold accumulation even of single nucleotides and at the same time trigger exponential replication [1]. Accumulation is driven by thermophoresis and convection in elongated chambers, replication by the inherent temperature cycling in convection. Optothermal pumping [2,3] allows to implement the thermal trap efficiently in a toroidal [4] or linear [5] geometry. Based on this method, we were in a position to combine accumulation and replication of DNA in the same chamber [5]. As we are missing a solid chemistry of prebiotic replication, we used as a proxy reaction for to replication the polymerase chain reaction. Convective flow both drives the DNA replicating polymerase chain reaction (PCR) while concurrent thermophoresis accumulates the replicated 143 base pair DNA in bulk solution. The time constant for accumulation is 92 s while DNA is doubled every 50 s. The length of the amplified DNA is checked with thermophoresis. Finite element simulations confirm the findings. The experiments explore conditions in pores of hydrothermal rock which can serve as a model environment for the origin of life and has prospects towards the first autonomous evolution, hosting the Darwin process by molecular selection using the thermophoretic trap. On the other side, the implemented continuous evolution will be able to breed well specified DNA or RNA molecules in the future. \\[4pt] [1] Baaske, Weinert, Duhr, Lemke, Russell and Braun, PNAS 104, 9346 (2007) \\[0pt] [2] Weinert, Kraus, Franosch and Braun, PRL 100, 164501 (2008) \\[0pt] [3] Weinert and Braun, Journal of Applied Physics 104, 104701 (2008) \\[0pt] [4] Weinert and Braun, Nano Letters 9, 4264 (2009) \\[0pt] [5] Mast and Braun, PRL 104, 188102 (2010) [Preview Abstract] |
Thursday, March 24, 2011 3:42PM - 4:18PM |
X7.00003: Single-molecule measurements of replisome composition and function reveal the mechanism of polymerase exchange Invited Speaker: A complete understanding of the molecular mechanisms underlying the functioning of large, multiprotein complexes requires experimental tools capable of simultaneously visualizing molecular architecture and enzymatic activity in real time. I will describe a novel single-molecule assay that combines the flow-stretching of individual DNA molecules to measure the activity of the DNA-replication machinery with the visualization of fluorescently labeled DNA polymerases at the replication fork. By correlating polymerase stoichiometry with DNA synthesis of T7 bacteriophage replisomes, we are able to quantitatively describe the mechanism of polymerase exchange. We find that even at relatively modest polymerase concentration (2 nM), soluble polymerases are recruited to an actively synthesizing replisome, dramatically increasing local polymerase concentration. These excess polymerases remain passively associated with the replisome through electrostatic interactions with the T7 helicase for 50 seconds until a stochastic and transient dissociation of the synthesizing polymerase from the primer-template allows for a polymerase exchange event to occur. [Preview Abstract] |
Thursday, March 24, 2011 4:18PM - 4:54PM |
X7.00004: Defects and DNA replication: a role for stochasticity Invited Speaker: When a cell replicates its DNA, each base must be copied once and only once per cell cycle. A failure to complete replication normally can lead to cell death, or worse. In this talk, I will discuss how ideas from statistical physics can help understand how replication is organized and controlled. In particular, we describe a formalism based on rate equations similar to those used to describe the kinetics of crystal growth and show how it can describe the normal course of DNA replication. In practice, replication must also deal with numerous kinds of problems. For example, the machinery of replication may stall or strands may be broken. We show how to extend our formalism to include the effects of such damage and conclude that there are two regimes: a normal regime, where the influence of defects is local, and an initiation-limited regime, where the influence of defects is long range. In the latter regime, defects have a global impact on replication. We show that normal, healthy cells have defect densities in the normal regime but cells with ``problems'' have defect densities that approach the crossover value. The overall conclusion is that passive stochastic control and physical effects such as diffusion are more relevant for DNA replication than had been believed until recently. [Preview Abstract] |
Thursday, March 24, 2011 4:54PM - 5:30PM |
X7.00005: Universal Temporal Profile of Replication Origin Activation in Eukaryotes Invited Speaker: The complete and faithful transmission of eukaryotic genome to daughter cells involves the timely duplication of mother cell's DNA. DNA replication starts at multiple chromosomal positions called replication origin. From each activated replication origin two replication forks progress in opposite direction and duplicate the mother cell's DNA. While it is widely accepted that in eukaryotic organisms replication origins are activated in a stochastic manner, little is known on the sources of the observed stochasticity. It is often associated to the population variability to enter S phase. We extract from a growing \textit{Saccharomyces cerevisiae} population the average rate of origin activation in a single cell by combining single molecule measurements and a numerical deconvolution technique. We show that the temporal profile of the rate of origin activation in a single cell is similar to the one extracted from a replicating cell population. Taking into account this observation we exclude the population variability as the origin of observed stochasticity in origin activation. We confirm that the rate of origin activation increases in the early stage of S phase and decreases at the latter stage. The population average activation rate extracted from single molecule analysis is in prefect accordance with the activation rate extracted from published micro-array data, confirming therefore the homogeneity and genome scale invariance of dynamic of replication process. All these observations point toward a possible role of replication fork to control the rate of origin activation. [Preview Abstract] |
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