SummaryIn higher eukaryotes, up to 70% of genes have high levels of nonmethylated cytosine/guanine base pairs (CpGs) surrounding promoters and gene regulatory units. These features, called CpG islands, were identified over 20 years ago, but there remains little mechanistic evidence to suggest how these enigmatic elements contribute to promoter function, except that they are refractory to epigenetic silencing by DNA methylation. Here we show that CpG islands directly recruit the H3K36-specific lysine demethylase enzyme KDM2A. Nucleation of KDM2A at these elements results in removal of H3K36 methylation, creating CpG island chromatin that is uniquely depleted of this modification. KDM2A utilizes a zinc finger CxxC (ZF-CxxC) domain that preferentially recognizes nonmethylated CpG DNA, and binding is blocked when the CpG DNA is methylated, thus constraining KDM2A to nonmethylated CpG islands. These data expose a straightforward mechanism through which KDM2A delineates a unique architecture that differentiates CpG island chromatin from bulk chromatin.

Efficient duplication of the genome requires the concerted action of helicase and DNA polymerases at replication forks1, to avoid stalling of the replication machinery and consequent genomic instability2-4. In eukaryotes, the physical coupling between helicase and DNA polymerases remains poorly understood. Here we define the molecular mechanism by which the yeast Ctf4 protein links the Cdc45-MCM-GINS (CMG) DNA helicase to DNA polymerase α (Pol α) within the replisome. We use X-ray crystallography and electron microscopy to show that Ctf4 self-associates in a constitutive disk-shaped trimer. Trimerization depends on a β-propeller domain in the carboxy-terminal half of the protein, which is fused to a helical extension that protrudes from one face of the trimeric disk. Critically, Pol α and the CMG helicase share a common mechanism of interaction with Ctf4. We show that the N-terminal tails of the catalytic subunit of Pol α and the Sld5 subunit of GINS contain a conserved Ctf4-binding motif that docks onto the exposed helical extension of a Ctf4 protomer within the trimer. Accordingly, we demonstrate that one Ctf4 trimer can support binding of up to three partner proteins, including the simultaneous association with both Pol α and GINS. Our findings indicate that Ctf4 can couple two molecules of Pol α to one CMG helicase within the replisome, providing a new paradigm for lagging-strand synthesis in eukaryotes that resembles the emerging model for the simpler replisome of E. coli5-8. The ability of Ctf4 to act as a platform for multivalent interactions illustrates a mechanism for the concurrent recruitment of factors that act together at the fork.

The Cdc45-MCM-GINS (CMG) helicase unwinds DNA during the elongation step of eukaryotic genome duplication and this process depends on the MCM ATPase function. Whether CMG translocation occurs on single- or double-stranded DNA and how ATP hydrolysis drives DNA unwinding remain open questions. Here we use cryo-electron microscopy to describe two subnanometre resolution structures of the CMG helicase trapped on a DNA fork. In the predominant state, the ring-shaped C-terminal ATPase of MCM is compact and contacts single-stranded DNA, via a set of pre-sensor 1 hairpins that spiral around the translocation substrate. In the second state, the ATPase module is relaxed and apparently substrate free, while DNA intimately contacts the downstream amino-terminal tier of the MCM motor ring. These results, supported by single-molecule FRET measurements, lead us to suggest a replication fork unwinding mechanism whereby the N-terminal and AAA+ tiers of the MCM work in concert to translocate on single-stranded DNA.

system (Zhongling Technology, Gihand) through linking between the somatosensory glove and robotic hand. The cryogenic environment was created by dry ice. For real-time energy delivery, SSANF was attached to a deformable robotic arm (Kinova, Jaco2) with luminescence LEDs connected with the output port. A power source with alternating voltage with an amplitude of 7.5 V was connected with the input port. The sizes of SSANFs used in the multifunctional robot system are all in sizes of 300 × 10 × 3.4 mm 3 .

The replisome unwinds and synthesizes DNA for genome duplication. In eukaryotes, the Cdc45-MCM-GINS (CMG) helicase and the leading-strand polymerase, Pol epsilon, form a stable assembly. The mechanism for coupling DNA unwinding with synthesis is starting to be elucidated, however the architecture and dynamics of the replication fork remain only partially understood, preventing a molecular understanding of chromosome replication. To address this issue, we conducted a systematic single-particle EM study on multiple permutations of the reconstituted CMG-Pol epsilon assembly. Pol epsilon contains two flexibly tethered lobes. The noncatalytic lobe is anchored to the motor of the helicase, whereas the polymerization domain extends toward the side of the helicase. We observe two alternate configurations of the DNA synthesis domain in the CMG-bound Pol epsilon. We propose that this conformational switch might control DNA template engagement and release, modulating replisome progression.DNA replication | CMG helicase | DNA polymerase | single-particle electron microscopy D NA replication is catalyzed by the replisome, a molecular machine that coordinates DNA unwinding and synthesis (1). These two functions must be tightly coordinated to prevent the rise of genome instability, which is a major cause of cancer. DNA unwinding by a replicative helicase involves single-strand translocation of a hexameric motor, whereas DNA synthesis requires template priming by a primase and extension by dedicated replicative DNA polymerases (2). In eukaryotes, the helicase function is performed by the Cdc45-MCM-GINS (CMG) complex (3, 4) and the primase function is played by Pol alpha (5), whereas DNA synthesis is catalyzed by two specialized DNA polymerases, Pol epsilon and delta. According to the consensus view, Pol epsilon synthesizes the leading and Pol delta the lagging strand (6-11). However, recent studies indicate that the division of labor between replicative polymerases might be more promiscuous than originally thought (12, 13). In in vitroreconstituted DNA replication reactions, Pol delta can support leading-strand duplication (11, 14), but switching from Pol delta to epsilon is necessary for efficient establishment of leadingstrand synthesis (14). The mechanism of substrate handoff between the two polymerases is currently unknown.Recent breakthroughs in structural biology begin to provide an architectural framework to understand the interaction between helicase and polymerases at the replication fork. For example, studies on the CMG helicase and its subcomplexes have established that the MCM is a six-member ring with an N-terminal domain that serves as a processivity collar (15) and a C-terminal ATPase motor domain that provides the DNA unwinding function (16-21). High-resolution cryo-EM analysis has shown that the ATPase motor translocates on the leading-strand template (22), in agreement with work on Xenopus embryo extracts (23). The GINS and Cdc45 components of the CMG bind to the side and stabilize the N-terminal domain of the...

We report a new facile strategy for quickly synthesizing pH sensitive poly(VI-co-HEA) hydrogels (VI ¼ N-vinylimidazole; HEA ¼ 2-hydroxyethyl acrylate) by frontal polymerization. The appropriate amounts of VI, HEA, and ammonium persulfate (APS)/N,N,N 0 ,N 0 -tetramethylethylenediamine (TMEDA) couple redox initiator were mixed together at ambient temperature in the presence of glycerol as the solvent medium. Frontal polymerization (FP) was initiated by heating the upper side of the mixture with a soldering iron. Once initiated, no further energy was required for the polymerization to occur. The dependence of the front velocity and front temperature on the VI/HEA weight ratios were investigated. The pH sensitive behavior, morphology, and heavy metal removal study of poly(VI-co-HEA) hydrogels prepared via FP were comparatively investigated on the basis of swelling measurements, scanning electron microscopy, and inductively coupling plasma spectrometer. Results show that the poly(VI-co-HEA) hydrogels prepared via FP exhibit good pH sensitivity and adsorption capacity. The FP can be exploited as an alternative means for synthesis of pH sensitive hydrogels in a fast and efficient way. The as-prepared hydrogels can be applied to remove heavy metals. V C 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: [4005][4006][4007][4008][4009][4010][4011][4012] 2010

We report a novel approach for quickly fabricating poly(NVP-co-NMA) hydrogels (NVP = 1-vinyl-2-pyrrolidone, NMA = N-methylolacrylamide) by using plasma-ignited frontal polymerization (PIFP). Frontal polymerization was initiated by igniting the upper side of the mixture with plasma. Once initiated, no additional energy was required for the polymerization to occur. We thoroughly investigated the influence of the molar ratios of NVP/NMA on the properties of poly(NVP-co-NMA) hydrogels via this PIFP approach. The swelling rate, swelling behavior, and morphology of polymer hydrogels prepared via PIFP were comparatively investigated on the basis of water contact angle, swelling measurement and scanning electron microscopy (SEM). Results show that the swelling property of poly(NVP-co-NMA) hydrogels depends on NVP/NMA molar ratios, and the swelling capacity of the hydrogels prepared using PIFP approach is superior to that obtained using traditional batch polymerization (BP) method. More interestingly, these hydrogels, able to absorb quantum dots (QDs), exhibit obvious switching effect via altering the process of swelling and deswelling, which can be developed as QD-based moisture sensor.

We describe a facile fabrication of white light‐emitting cadmium sulfide (CdS)‐poly(HEA‐co‐NVK) nanocomposites [2‐hydroxyethyl acrylate (HEA) and N‐vinylcarbazole (NVK)] via plasma‐ignited frontal polymerization (PIFP), a novel and rapid reaction mode of converting monomers into polymers in minutes. Frontal polymerization was initiated by igniting the upper side of the reactant with plasma. Once initiated, no additional energy was required for the polymerization to occur. The chemical functional groups of the as‐prepared nanocomposites were thoroughly investigated using Fourier transform infrared spectra. The dependence of the front velocity and front temperature on the initiator concentration and weight ratios of HEA/NVK was also investigated in detail. Perhaps more interestingly, the white light‐emitting materials synthesized by ingeniously incorporating the compensating colors of yellow emitting from 3‐(trimethoxysilyl)‐1‐propanethiol‐capped CdS nanocrystals and blue emitting from carbazole‐containing polymer were conveniently applied onto a commercial UV light‐emitting diode (LED) to generate white LEDs. The subtle change in the weight ratios of CdS/NVK can significantly impact the color hue. The white light becomes gradually colder with the increase of NVK, but becomes gradually warmer with the increase concentration of CdS nanocrystals. In a broad perspective, these white light‐emitting materials designed by PIFP approach will open a new pathway to develop “QD‐polymer nanocomposite down‐conversion LED” in a fast and efficient way. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2012