Distance dependence of fluorescence resonance energy transfer

Deviations from the usual R (-6) dependence of the rate of fluorescence resonance energy transfer (FRET) on the distance between the donor and the acceptor have been a common scenario in the recent times. In this paper, we present a critical analysis of the distance dependence of FRET, and try to illustrate the non R (-6) type behaviour of the rate for the case of transfer from a localized electronic excitation on the donor, a dye molecule to three different energy acceptors with delocalized electronic excitations namely, graphene,two-dimensional semiconducting sheet and the case of such a semiconducting sheet rolled to obtain a nanotube. We use simple analytic models to understand the distance dependence in each case.

Single-pair fluorescence resonance energy transfer on freely diffusing molecules: Observation of Förster distance dependence and subpopulations

Photon bursts from single diffusing donor-acceptor labeled macromolecules were used to measure intramolecular distances and identify subpopulations of freely diffusing macromolecules in a heterogeneous ensemble. By using DNA as a rigid spacer, a series of constructs with varying intramolecular donor-acceptor spacings were used to measure the mean and distribution width of fluorescence resonance energy transfer (FRET) efficiencies as a function of distance. The mean single-pair FRET efficiencies qualitatively follow the distance dependence predicted by Förster theory. Possible contributions to the widths of the FRET efficiency distributions are discussed, and potential applications in the study of biopolymer conformational dynamics are suggested. The ability to measure intramolecular (and intermolecular) distances for single molecules implies the ability to distinguish and monitor subpopulations of molecules in a mixture with different distances or conformational states. This is demonstrated by monitoring substrate and product subpopulations before and after a restriction endonuclease cleavage reaction. Distance measurements at single-molecule resolution also should facilitate the study of complex reactions such as biopolymer folding. To this end, the denaturation of a DNA hairpin was examined by using single-pair FRET.

Non-monotonic, distance-dependent relaxation of water in reverse micelles: Propagation of surface induced frustration along hydrogen bond networks

Layer-wise, distance-dependent orientational relaxation of water confined in reverse micelles (RM) is studied using theoretical and computational tools. We use both a newly constructed ``spins on a ring'' (SOR) Ising-type model (with Shore-Zwanzig rotational dynamics) and atomistic simulations with explicit water. Our study explores the effect of reverse micelle size and role of intermolecular correlations, compromised by the presence of a highly polar surface, on the distance (from the interface) dependence of water relaxation. The ``spins on a ring'' model can capture some aspects of distance dependence of relaxation, such as acceleration of orientational relaxation at intermediate layers. In atomistic simulations, layer-wise decomposition of hydrogen bond formation pattern clearly reveals that hydrogen bond arrangement of water at a certain distance away from the surface can remain frustrated due to the interaction with the polar surface head groups. This layer-wise analysis also reveals the presence of a non-monotonic slow relaxation component which can be attributed to this frustration effect and which is accentuated in small to intermediate size RMs. For large size RMs, the long time component decreases monotonically from the interface to the interior of the RMs with slowest relaxation observed at the interface. (C) 2012 American Institute of Physics. http://dx.doi.org/10.1063/1.4732095]

Generalized survival in step fluctuations

The properties of the generalized survival probability, that is, the probability of not crossing an arbitrary location R during relaxation, have been investigated experimentally (via scanning tunneling microscope observations) and numerically. The results confirm that the generalized survival probability decays exponentially with a time constant tau(s)(R). The distance dependence of the time constant is shown to be tau(s)(R)=tau(s0)exp[-R/w(T)], where w(2)(T) is the material-dependent mean-squared width of the step fluctuations. The result reveals the dependence on the physical parameters of the system inherent in the prior prediction of the time constant scaling with R/L-alpha, with L the system size and alpha the roughness exponent. The survival behavior is also analyzed using a contrasting concept, the generalized inside survival S-in(t,R), which involves fluctuations to an arbitrary location R further from the average. Numerical simulations of the inside survival probability also show an exponential time dependence, and the extracted time constant empirically shows (R/w)(lambda) behavior, with lambda varying over 0.6 to 0.8 as the sampling conditions are changed. The experimental data show similar behavior, and can be well fit with lambda=1.0 for T=300 K, and 0.5

Excitation energy transfer from dye molecules to doped graphene

Recently, we have reported theoretical studies on the rate of energy transfer from an electronically excited molecule to graphene. It was found that graphene is a very efficient quencher of the electronically excited states and that the rate infinity z(-4). The process was found to be effective up to 30 nm which is well beyond the traditional FRET limit. In this report, we study the transfer of an amount of energy (h) over bar Omega from a dye molecule to doped graphene. We find a crossover of the distance dependence of the rate from z(-4) to exponential as the Fermi level is increasingly shifted into the conduction band, with the crossover occurring at a shift of the Fermi level by an amount (h) over bar Omega/2.

Sensitivity of polarization fluctuations to the nature of protein-water interactions: Study of biological water in four different protein-water systems

Since the time of Kirkwood, observed deviations in magnitude of the dielectric constant of aqueous protein solution from that of neat water (similar to 80) and slower decay of polarization have been subjects of enormous interest, controversy, and debate. Most of the common proteins have large permanent dipole moments (often more than 100 D) that can influence structure and dynamics of even distant water molecules, thereby affecting collective polarization fluctuation of the solution, which in turn can significantly alter solution's dielectric constant. Therefore, distance dependence of polarization fluctuation can provide important insight into the nature of biological water. We explore these aspects by studying aqueous solutions of four different proteins of different characteristics and varying sizes, chicken villin headpiece subdomain (HP-36), immunoglobulin binding domain protein G (GB1), hen-egg white lysozyme (LYS), and Myoglobin (MYO). We simulate fairly large systems consisting of single protein molecule and 20000-30000 water molecules (varied according to the protein size), providing a concentration in the range of similar to 2-3 mM. We find that the calculated dielectric constant of the system shows a noticeable increment in all the cases compared to that of neat water. Total dipole moment auto time correlation function of water < dM(W) (0)delta M-W (t) > is found to be sensitive to the nature of the protein. Surprisingly, dipole moment of the protein and total dipole moment of the water molecules are found to be only weakly coupled. Shellwise decomposition of water molecules around protein reveals higher density of first layer compared to the succeeding ones. We also calculate heuristic effective dielectric constant of successive layers and find that the layer adjacent to protein has much lower value (similar to 50). However, progressive layers exhibit successive increment of dielectric constant, finally reaching a value close to that of bulk 4-5 layers away. We also calculate shellwise orientational correlation function and tetrahedral order parameter to understand the local dynamics and structural re-arrangement of water. Theoretical analysis providing simple method for calculation of shellwise local dielectric constant and implication of these findings are elaborately discussed in the present work. (C) 2014 AIP Publishing LLC.

Orientational order as the origin of the long-range hydrophobic effect

The long range attractive force between two hydrophobic surfaces immersed in water is observed to decrease exponentially with their separation-this distance-dependence of effective force is known as the hydrophobic force law (HFL). We explore the microscopic origin of HFL by studying distance-dependent attraction between two parallel rods immersed in 2D Mercedes Benz model of water. This model is found to exhibit a well-defined HFL. Although the phenomenon is conventionally explained by density-dependent theories, we identify orientation, rather than density, as the relevant order parameter. The range of density variation is noticeably shorter than that of orientational heterogeneity. The latter is comparable to the observed distances of hydrophobic force. At large separation, attraction between the rods arises primarily from a destructive interference among the inwardly propagating oppositely oriented heterogeneity generated in water by the two rods. As the rods are brought closer, the interference increases leading to a decrease in heterogeneity and concomitant decrease in free energy of the system, giving rise to the effective attraction. We notice formation of hexagonal ice-like structures at the onset of attractive region which suggests that metastable free energy minimum may play a role in the origin of HFL. (C) 2015 AIP Publishing LLC.

Electron tunneling in proteins and water

The subject of this thesis is electronic coupling in donor-bridge-acceptor systems. In Chapter 2, ET properties of cyanide-bridged dinuclear ruthenium complexes were investigated. The strong interaction between the mixed-valent ruthenium centers leads to intense metal-to-metal charge transfer bands (MMCT). Hush analysis of the MMCT absorption bands yields the electronic-coupling strength between the metal centers (H_(AB)) and the total reorganization energy (λ). Comparison of ET kinetics to calculated rates shows that classical ET models fail to account for the observed kinetics and nuclear tunneling must be considered.

In Chapter 3, ET rates were measured in four ruthenium-modified highpotential iron-sulfur proteins (HiPIP), which were modified at position His50, His81, His42 and His18, respectively. ET kinetics for the His50 and His81 mutants are a factor of 300 different, while the donor-acceptor separation is nearly identical. PATHWAY calculations corroborate these measurements and highlight the importance of structural detail of the intervening protein matrix.

In Chapter 4, the distance dependence of ET through water bridges was measured. Photoinduced ET measurements in aqueous glasses at 77 K show that water is a poor medium for ET. Luminescence decay and quantum yield data were analyzed in the context of a quenching model that accounts for the exponential distance dependence of ET, the distance distribution of donors and acceptors embedded in the glass and the excluded volumes generated by the finite sizes of the donors and acceptors.

In Chapter 5, the pH-dependent excited state dynamics of ruthenium-modified amino acids were measured. The [Ru(bpy)_(3)] ^(2+) chromophore was linked to amino acids via an amide linkage. Protonation of the amide oxygen effectively quenches the excited state. In addition. time-resolved and steady-state luminescence data reveal that nonradiative rates are very sensitive to the protonation state and the structure of the amino acid moiety.

DNA-mediated oxidation of transcription factor p53

Transcription factor p53 is the most commonly altered gene in human cancer. As a redox-active protein in direct contact with DNA, p53 can directly sense oxidative stress through DNA-mediated charge transport. Electron hole transport occurs with a shallow distance dependence over long distances through the π-stacked DNA bases, leading to the oxidation and dissociation of DNA-bound p53. The extent of p53 dissociation depends upon the redox potential of the response element DNA in direct contact with each p53 monomer. The DNA sequence dependence of p53 oxidative dissociation was examined by electrophoretic mobility shift assays using radiolabeled oligonucleotides containing both synthetic and human p53 response elements with an appended anthraquinone photooxidant. Greater p53 dissociation is observed from DNA sequences containing low redox potential purine regions, particularly guanine triplets, within the p53 response element. Using denaturing polyacrylamide gel electrophoresis of irradiated anthraquinone-modified DNA, the DNA damage sites, which correspond to locations of preferred electron hole localization, were determined. The resulting DNA damage preferentially localizes to guanine doublets and triplets within the response element. Oxidative DNA damage is inhibited in the presence of p53, however, only at DNA sites within the response element, and therefore in direct contact with p53. From these data, predictions about the sensitivity of human p53-binding sites to oxidative stress, as well as possible biological implications, have been made. On the basis of our data, the guanine pattern within the purine region of each p53-binding site determines the response of p53 to DNA-mediated oxidation, yielding for some sequences the oxidative dissociation of p53 from a distance and thereby providing another potential role for DNA charge transport chemistry within the cell.

To determine whether the change in p53 response element occupancy observed in vitro also correlates in cellulo, chromatin immunoprecipition (ChIP) and quantitative PCR (qPCR) were used to directly quantify p53 binding to certain response elements in HCT116N cells. The HCT116N cells containing a wild type p53 were treated with the photooxidant [Rh(phi)2bpy]³⁺, Nutlin-3 to upregulate p53, and subsequently irradiated to induce oxidative genomic stress. To covalently tether p53 interacting with DNA, the cells were fixed with disuccinimidyl glutarate and formaldehyde. The nuclei of the harvested cells were isolated, sonicated, and immunoprecipitated using magnetic beads conjugated with a monoclonal p53 antibody. The purified immounoprecipiated DNA was then quantified via qPCR and genomic sequencing. Overall, the ChIP results were significantly varied over ten experimental trials, but one trend is observed overall: greater variation of p53 occupancy is observed in response elements from which oxidative dissociation would be expected, while significantly less change in p53 occupancy occurs for response elements from which oxidative dissociation would not be anticipated.

The chemical oxidation of transcription factor p53 via DNA CT was also investigated with respect to the protein at the amino acid level. Transcription factor p53 plays a critical role in the cellular response to stress stimuli, which may be modulated through the redox modulation of conserved cysteine residues within the DNA-binding domain. Residues within p53 that enable oxidative dissociation are herein investigated. Of the 8 mutants studied by electrophoretic mobility shift assay (EMSA), only the C275S mutation significantly decreased the protein affinity (KD) for the Gadd45 response element. EMSA assays of p53 oxidative dissociation promoted by photoexcitation of anthraquinone-tethered Gadd45 oligonucleotides were used to determine the influence of p53 mutations on oxidative dissociation; mutation to C275S severely attenuates oxidative dissociation while C277S substantially attenuates dissociation. Differential thiol labeling was used to determine the oxidation states of cysteine residues within p53 after DNA-mediated oxidation. Reduced cysteines were iodoacetamide labeled, while oxidized cysteines participating in disulfide bonds were ¹³C₂D₂-iodoacetamide labeled. Intensities of respective iodoacetamide-modified peptide fragments were analyzed using a QTRAP 6500 LC-MS/MS system, quantified with Skyline, and directly compared. A distinct shift in peptide labeling toward ¹³C₂D₂-iodoacetamide labeled cysteines is observed in oxidized samples as compared to the respective controls. All of the observable cysteine residues trend toward the heavy label under conditions of DNA CT, indicating the formation of multiple disulfide bonds potentially among the C124, C135, C141, C182, C275, and C277. Based on these data it is proposed that disulfide formation involving C275 is critical for inducing oxidative dissociation of p53 from DNA.

Investigations of DNA-mediated protein oxidation

DNA charge transport (CT) involves the efficient transfer of electrons or electron holes through the DNA π-stack over long molecular distances of at least 100 base-pairs. Despite this shallow distance dependence, DNA CT is sensitive to mismatches or lesions that disrupt π-stacking and is critically dependent on proper electronic coupling of the donor and acceptor moieties into the base stack. Favorable DNA CT is very rapid, occurring on the picosecond timescale. Because of this speed, electron holes equilibrate along the DNA π-stack, forming a characteristic pattern of DNA damage at low oxidation potential guanine multiplets. Furthermore, DNA CT may be used in a biological context. DNA processing enzymes with 4Fe4S clusters can perform DNA-mediated electron transfer (ET) self-exchange reactions with other 4Fe4S cluster proteins, even if the proteins are quite dissimilar, as long as the DNA-bound [4Fe4S]^3+/2+ redox potentials are conserved. This mechanism would allow low copy number DNA repair proteins to find their lesions efficiently within the cell. DNA CT may also be used biologically for the long-range, selective activation of redox-active transcription factors. Within this work, we pursue other proteins that may utilize DNA CT within the cell and further elucidate aspects of the DNA-mediated ET self-exchange reaction of 4Fe4S cluster proteins.

Dps proteins, bacterial mini-ferritins that protect DNA from oxidative stress, are implicated in the survival and virulence of pathogenic bacteria. One aspect of their protection involves ferroxidase activity, whereby ferrous iron is bound and oxidized selectively by hydrogen peroxide, thereby preventing formation of damaging hydroxyl radicals via Fenton chemistry. Understanding the specific mechanism by which Dps proteins protect the bacterial genome could inform the development of new antibiotics. We investigate whether DNA-binding E. coli Dps can utilize DNA CT to protect the genome from a distance. An intercalating ruthenium photooxidant was employed to generate oxidative DNA damage via the flash-quench technique, which localizes to a low potential guanine triplet. We find that Dps loaded with ferrous iron, in contrast to Apo-Dps and ferric iron-loaded Dps which lack available reducing equivalents, significantly attenuates the yield of oxidative DNA damage at the guanine triplet. These data demonstrate that ferrous iron-loaded Dps is selectively oxidized to fill guanine radical holes, thereby restoring the integrity of the DNA. Luminescence studies indicate no direct interaction between the ruthenium photooxidant and Dps, supporting the DNA-mediated oxidation of ferrous iron-loaded Dps. Thus DNA CT may be a mechanism by which Dps efficiently protects the genome of pathogenic bacteria from a distance.

Further work focused on spectroscopic characterization of the DNA-mediated oxidation of ferrous iron-loaded Dps. X-band EPR was used to monitor the oxidation of DNA-bound Dps after DNA photooxidation via the flash-quench technique. Upon irradiation with poly(dGdC)₂, a signal arises with g = 4.3, consistent with the formation of mononuclear high-spin Fe(III) sites of low symmetry, the expected oxidation product of Dps with one iron bound at each ferroxidase site. When poly(dGdC)₂ is substituted with poly(dAdT)₂, the yield of Dps oxidation is decreased significantly, indicating that guanine radicals facilitate Dps oxidation. The more favorable oxidation of Dps by guanine radicals supports the feasibility of a long-distance protection mechanism via DNA CT where Dps is oxidized to fill guanine radical holes in the bacterial genome produced by reactive oxygen species.

We have also explored possible electron transfer intermediates in the DNA-mediated oxidation of ferrous iron-loaded Dps. Dps proteins contain a conserved tryptophan residue in close proximity to the ferroxidase site (W52 in E. coli Dps). In comparison to WT Dps, in EPR studies of the oxidation of ferrous iron-loaded Dps following DNA photooxidation, W52Y and W52A mutants were deficient in forming the characteristic EPR signal at g = 4.3, with a larger deficiency for W52A compared to W52Y. In addition to EPR, we also probed the role of W52 Dps in cells using a hydrogen peroxide survival assay. Bacteria containing W52Y Dps survived the hydrogen peroxide challenge more similarly to those containing WT Dps, whereas cells with W52A Dps died off as quickly as cells without Dps. Overall, these results suggest the possibility of W52 as a CT hopping intermediate.

DNA-modified electrodes have become an essential tool for the study of the redox chemistry of DNA processing enzymes with 4Fe4S clusters. In many cases, it is necessary to investigate different complex samples and substrates in parallel in order to elucidate this chemistry. Therefore, we optimized and characterized a multiplexed electrochemical platform with the 4Fe4S cluster base excision repair glycosylase Endonuclease III (EndoIII). Closely packed DNA films, where the protein has limited surface accessibility, produce EndoIII electrochemical signals sensitive to an intervening mismatch, indicating a DNA-mediated process. Multiplexed analysis allowed more robust characterization of the CT-deficient Y82A EndoIII mutant, as well as comparison of a new family of mutations altering the electrostatics surrounding the 4Fe4S cluster in an effort to shift the reduction potential of the cluster. While little change in the DNA-bound midpoint potential was found for this family of mutants, likely indicating the dominant effect of DNA-binding on establishing the protein redox potential, significant variations in the efficiency of DNA-mediated electron transfer were apparent. On the basis of the stability of these proteins, examined by circular dichroism, we proposed that the electron transfer pathway in EndoIII can be perturbed not only by the removal of aromatic residues but also through changes in solvation near the cluster.

While the 4Fe4S cluster of EndoIII is relatively insensitive to oxidation and reduction in solution, we have found that upon DNA binding, the reduction potential of the [4Fe4S]^3+/2+ couple shifts negatively by approximately 200 mV, bringing this couple into a physiologically relevant range. Demonstrated using electrochemistry experiments in the presence and absence of DNA, these studies do not provide direct molecular evidence for the species being observed. Sulfur K-edge X-ray absorbance spectroscopy (XAS) can be used to probe directly the covalency of iron-sulfur clusters, which is correlated to their reduction potential. We have shown that the Fe-S covalency of the 4Fe4S cluster of EndoIII increases upon DNA binding, stabilizing the oxidized [4Fe4S]³⁺ cluster, consistent with a negative shift in reduction potential. The 7% increase in Fe-S covalency corresponds to an approximately 150 mV shift, remarkably similar to DNA electrochemistry results. Therefore we have obtained direct molecular evidence for the shift in 4Fe4S reduction potential of EndoIII upon DNA binding, supporting the feasibility of our model whereby these proteins can utilize DNA CT to cooperate in order to efficiently find DNA lesions inside cells.

In conclusion, in this work we have explored the biological applications of DNA CT. We discovered that the DNA-binding bacterial ferritin Dps can protect the bacterial genome from a distance via DNA CT, perhaps contributing to pathogen survival and virulence. Furthermore, we optimized a multiplexed electrochemical platform for the study of the redox chemistry of DNA-bound 4Fe4S cluster proteins. Finally, we have used sulfur K-edge XAS to obtain direct molecular evidence for the negative shift in 4Fe4S cluster reduction potential of EndoIII upon DNA binding. These studies contribute to the understanding of DNA-mediated protein oxidation within cells.

DNA-mediated charge transport signaling within the cell

DNA possesses the curious ability to conduct charge longitudinally through the π-stacked base pairs that reside within the interior of the double helix. The rate of charge transport (CT) through DNA has a shallow distance dependence. DNA CT can occur over at least 34 nm, a very long molecular distance. Lastly, DNA CT is exquisitely sensitive to disruptions, such as DNA damage, that affect the dynamics of base-pair stacking. Many DNA repair and DNA-processing enzymes are being found to contain 4Fe-4S clusters. These co-factors have been found in glycosylases, helicases, helicase-nucleases, and even enzymes such as DNA polymerase, RNA polymerase, and primase across the phylogeny. The role of these clusters in these enzymes has remained elusive. Generally, iron-sulfur clusters serve redox roles in nature since, formally, the cluster can exist in multiple oxidation states that can be accessed within a biological context. Taken together, these facts were used as a foundation for the hypothesis that DNA-binding proteins with 4Fe-4S clusters utilize DNA-mediated CT as a means to signal one another to scan the genome as a first step in locating the subtle damage that occurs within a sea of undamaged bases within cells.

Herein we describe a role for 4Fe-4S clusters in DNA-mediated charge transport signaling among EndoIII, MutY, and DinG, which are from distinct repair pathways in E. coli. The DinG helicase is an ATP-dependent helicase that contains a 4Fe-4S cluster. To study the DNA-bound redox properties of DinG, DNA-modified electrochemistry was used to show that the 4Fe-4S cluster of DNA-bound DinG is redox-active at cellular potentials, and shares the 80 mV vs. NHE redox potential of EndoIII and MutY. ATP hydrolysis by DinG increases the DNA-mediated redox signal observed electrochemically, likely reflecting better coupling of the 4Fe-4S cluster to DNA while DinG unwinds DNA, which could have interesting biological implications. Atomic force microscopy experiments demonstrate that DinG and EndoIII cooperate at long range using DNA charge transport to redistribute to regions of DNA damage. Genetics experiments, moreover, reveal that this DNA-mediated signaling among proteins also occurs within the cell and, remarkably, is required for cellular viability under conditions of stress. Knocking out DinG in CC104 cells leads to a decrease in MutY activity that is rescued by EndoIII D138A, but not EndoIII Y82A. DinG, thus, appears to help MutY find its substrate using DNA-mediated CT, but do MutY or EndoIII aid DinG in a similar way? The InvA strain of bacteria was used to observe DinG activity, since DinG activity is required within InvA to maintain normal growth. Silencing the gene encoding EndoIII in InvA results in a significant growth defect that is rescued by the overexpression of RNAseH, a protein that dismantles the substrate of DinG, R-loops. This establishes signaling between DinG and EndoIII. Furthermore, rescue of this growth defect by the expression of EndoIII D138A, the catalytically inactive but CT-proficient mutant of EndoIII, is also observed, but expression of EndoIII Y82A, which is CT-deficient but enzymatically active, does not rescue growth. These results provide strong evidence that DinG and EndoIII utilize DNA-mediated signaling to process DNA damage. This work thus expands the scope of DNA-mediated signaling within the cell, as it indicates that DNA-mediated signaling facilitates the activities of DNA repair enzymes across the genome, even for proteins from distinct repair pathways.

In separate work presented here, it is shown that the UvrC protein from E. coli contains a hitherto undiscovered 4Fe-4S cluster. A broad shoulder at 410 nm, characteristic of 4Fe-4S clusters, is observed in the UV-visible absorbance spectrum of UvrC. Electron paramagnetic resonance spectroscopy of UvrC incubated with sodium dithionite, reveals a spectrum with the signature features of a reduced, [4Fe-4S]+1, cluster. DNA-modified electrodes were used to show that UvrC has the same DNA-bound redox potential, of ~80 mV vs. NHE, as EndoIII, DinG, and MutY. Again, this means that these proteins are capable of performing inter-protein electron transfer reactions. Does UvrC use DNA-mediated signaling to facilitate the repair of its substrates?

UvrC is part of the nucleotide excision repair (NER) pathway in E. coli and is the protein within the pathway that performs the chemistry required to repair bulky DNA lesions, such as cyclopyrimidine dimers, that form as a product of UV irradiation. We tested if UvrC utilizes DNA-mediated signaling to facilitate the efficient repair of UV-induced DNA damage products by helping UvrC locate DNA damage. The UV sensitivity of E. coli cells lacking DinG, a putative signaling partner of UvrC, was examined. Knocking out DinG in E. coli leads to a sensitivity of the cells to UV irradiation. A 5-10 fold reduction in the amount of cells that survive after irradiation with 90 J/m2 of UV light is observed. This is consistent with the hypothesis that UvrC and DinG are signaling partners, but is this signaling due to DNA-mediated CT? Complementing the knockout cells with EndoIII D138A, which can also serve as a DNA CT signaling partner, rescues cells lacking DinG from UV irradiation, while complementing the cells with EndoIII Y82A shows no rescue of viability. These results indicate that there is cross-talk between the NER pathway and DinG via DNA-mediated signaling. Perhaps more importantly, this work also establishes that DinG, EndoIII, MutY, and UvrC comprise a signaling network that seems to be unified by the ability of these proteins to perform long range DNA-mediated CT signaling via their 4Fe-4S clusters.

古田山亚热带常绿阔叶林密度制约普遍性研究

密度制约是否为自然森林维持物种共存的普遍性机制，生态学家对此一直就有争议。目前密度制约的普遍性研究主要集中于热带森林，而针对亚热带森林的研究还比较少见。本文以浙江古田山24 公顷亚热带常绿阔叶林固定监测样地第一次调查数据为基础，主要采用点格局分析的双变量函数g(r)，研究了密度制约是否作用于样地内大部分木本植物，目的在于探讨密度制约在亚热带常绿阔叶林内物种多样性维持中的作用。 检验密度制约效应的常用方法是: 假定在种内竞争、种内个体之间的病虫害传播等条件下，种群的聚集程度是否随年龄的增加而下降。但是生境的异质性也可能导致种群聚集程度的下降。所以在密度制约的普遍性检验之前，首先分析了生境异质性是否影响树木的分布，然后分析种群分布格局，探讨除了生境异质性以外其它影响种群分布的驱动因子，同时为分析密度制约的发生做解释。结果发现生境异质性影响古田山树木的分布。用完全随机零模型不排除异质性，被检验的64个物种几乎在0-30m所有的尺度上都表现聚集。用异质性泊松分布零模型排除异质性，59个被检验物种中58个表现聚集。排不排除异质性，同种聚集都在整个样地中占主要地位，而且随着远离目标个体同种个体的密度逐渐下降，植株主要聚集在同种邻体的周围。结合下面的关联性分析，更新植株主要集中在成年个体周围，说明除了生境异质性效应促进树木聚集分布以外，以繁殖体为中心的局部扩散是大部分物种同种聚集的主要原因。 环境异质性影响树木分布，干扰密度制约的检验。然而，排除生境异质性的影响，也不能肯定密度制约是否是群落物种多样性普遍性的维持机制。用随机标签零模型案例-对照设计，小径级生长阶段的树木格局作为案例，成年树作为对照代表生境异质性的作用，通过小径级树木格局与成年树格局相比，排除生境异质性的干扰，64个被检验物种中50种(78.1%)表现了密度制约稀疏效应。结果表明密度制约稀疏机制调节了样地大部分物种，是古田山亚热带森林群落物种多样性维持的重要机制。同时，密度制约稀疏效应主要发生在局部尺度上，与同种短距离聚集结果一致。另外，密度制约稀疏效应更易于影响丰富种(24公顷样地内个体数>1000)的种群结构。 Janzen-Connell假说的距离制约模型认为繁殖体制约后代更新成功，导致成年树个体间距增大，并得到众多的野外观察证明。然而，在大尺度的森林样地研究中，没有发现这个效应广泛存在。本研究采用独立性零模型分析了不同生活史阶段在空间上的关联性，特别是成年树和幼树、小树的关联性，反过来推演是否成年树对后代的距离制约驱动了种群分布的空间动态。综合分析古田山64个物种的不同径级阶段的空间关联性，同时参照Condit 等(1992，1994)的研究结果：成年树对后代的距离制约效应在小于5m的尺度上发生强烈。有20个物种（31.3%）的更新体聚集密度最高点在离成年树≥5m的距离上，加上21个物种的更新体与成年树的关联性成相互排斥和随机分布，64个被检验的物种中有41(64.1%)个物种表现了Janzen-Connell 假说的繁殖体对后代的距离制约效应。同时，54(84.4%)个物种的小树成为新的成年树，出现在成年体周围小于5m的距离内的比例最高。结果表明这个效应提高成年树的间距是有限的，然而，它仍然调节了样地内大部分物种的分布格局，促进了物种共存。 同种密度制约调节建成树木（胸径≥1cm）的空间结构得到很好的研究。然而最近的研究表明：病菌不仅在同种个体之间传播，同时也在近缘物种之间传播。因此，只包括同种个体的密度制约模型可能大大低估了密度制约效应的作用。本研究分析了古田山24 公顷内159 个物种。运用平均谱系多样性指数(APd) 和最近分类谱系多样性指数(NTPd)检验随着目标个体径级的增加系统发育结构变化的趋势。研究发现，在15、20、30m 的尺度上，APd 指数都随着生长阶段的增加而显著提高，在10、15、20、30m 的尺度上，NTPd 随着生长阶段的增加而显著提高；在5m 的尺度上，谱系多样性与生长阶段不存在显著关联性，这些结果表明谱系多样性制约与取样尺度相关。在DBH >30 cm 生长阶段， APd 下降，NTPd 上升，说明谱系多样性制约加大老树之间的谱系距离，但是由于类似的生境偏好，又倾向于聚集在类似的生境。 本研究的结果表明同种和近缘种的密度制约是亚热带常绿阔叶林生物多样性群落水平上重要的维持机制，为Janzen-Connell 假说提供了支持；同时，生境异质性和局部扩散能在维持森林物种多样性中发挥重要作用。

Effects of phase-breaking on long-range charge transfer in DNA: Partially-coherent-tunneling model study

The mechanism of hole charge transfer in DNA of various lengths and sequences is investigated based on a partially coherent tunneling theory (Zhang et al., J Chem Phys 117:4578, 2002), where the effects of phase-breaking in adenine-thymine and guanine-cytosine base pairs are treated on equal foot. This work aims at providing a self-consistent microscopic interpretation for rate experiments on various DNA systems. We will also clarify the condition under which the simple superexchange-mediated-hopping picture is valid, and make some comments on the further development of present theory.

A partially incoherent rate theory of long-range charge transfer in deoxyribose nucleic acid

A quantum chemistry based Green's function formulation of long-range charge transfer in deoxyribose nucleic acid (DNA) double helix is proposed. The theory takes into account the effects of DNA's electronic structure and its incoherent interaction with aqueous surroundings. In the implementation, the electronic tight-binding parameters for unsolvated DNA molecules are determined at the HF/6-31G* level, while those for individual nucleobase-water couplings are at a semiempirical level by fitting with experimental redox potentials. Numerical results include that: (i) the oxidative charge initially at the donor guanine site does hop sequentially over all guanine sites; however, the revealed rates can be of a much weaker distance dependence than that described by the ordinary Ohm's law; (ii) the aqueous surroundings-induced partial incoherences in thymine/adenine bridge bases lead them to deviate substantially from the superexchange regime; (iii) the time scale of the partially incoherent hole transport through the thymine/adenine pi stack in DNA is about 5 ps. (C) 2002 American Institute of Physics.

Effect of DNA flanking sequence on charge transport in short DNA duplexes

Several factors can influence charge transport (CT)-mediated DNA, such as sequence, distance, base stacking, base pair mismatch, conformation, tether length, etc. However, the DNA context effect or how flanking sequences influence redox active drugs in the DNA CT reaction and later in DNA enzymatic repair and synthesis is still not well understood. The set of seven DNA molecules in this study have been characterized well for the study of flanking sequence effects. These DNA duplexes are formed from self-complementary strands and contain the common central four-base sequence 5'-A-G-C-T-3', flanked on both sides by either (AT)(n) or (AA)(n) (n = 2, 3, or 4) or AA(AT)(2). UV-vis, fluorescence, UV melting, circular dichroism, and cyclic voltammetry experiments were used to study the flanking sequence effect on CT-mediated DNA by using daunomycin or adriamycin cross-linked with these seven DNA molecules. Our results showed that charge transport was related to the flanking sequence, DNA melting free energy, and ionic strength. For (AA)(n) or (AT)(n) species of the same length, (AA)(n) series were more stable and more efficient CT was observed through the (AA)(n) series. The same trend was observed for (AA)(n) and (AT)(n) series at different ionic strengths, further supporting the idea that flanking sequence can result in different base stacking and modulate charge transport through these seven DNA molecules.