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Traditional topics such as physics, chemistry and mathematics have immensely changed the world in the twentieth century, but the twenty-first century seems to be that of soft condensed matter physics, which has already shown its tremendous possibilities to influence the everyday human life through its technological manifestations such as biotechnology and soft nano technology. Unlike the traditional topics, soft condensed matter physics has an interdisciplinary nature. It studies systems that usually come under chemistry or biology, using the methods of physics and mathematics and hence, transcends the frontiers between the subjects. Soft matter may be classified into three main classes; colloidal dispersions, polymers and polymer melts and liquid crystals. Study of single polymer chains is a fascinating topic that provides insights to understand many processes occurring in biological systems. Here, we present analytical studies of a few such processes, involving single polymer chains. In fact, there are a number of biological processes, which involve the dynamics of a single polymer chain. Due to the importance of Brownian motion at the mesoscopic level, soft matter systems are always studied using the analytical as well as computational methods of statistical mechanics. The statistical mechanics of polymers has been developed into a fascinating topic due to the contributions from the theory of random walks and path integrals. The dynamical behavior of many-particle systems has been described traditionally by the so-called rate theories. Here, we use these classical approaches to study a few biological processes that involve single polymer chains. The kind of processes that we have investigated may be categorized into two, namely the processes that leads to conformational changes in a chain molecule and the processes involving spatial translocation of a polymer. In the first category, we have considered the dynamics of semiflexible polymer loops. Loop formation of chain molecules has a key role in biological processes like DNA replication, gene regulation and protein folding. Hence, the dynamics of a polymer closing to form a loop as well as opening of the loop are topics of considerable theoretical/experimental interest. For closing, results are available in the completely flexible limit. Wilemski and Fixman, (J. Chem. Phys.60, 878 (1974)) have studied the closing and opening reactions in a single flexible polymer chain and using their approach Doi found the closing time to vary with the length of the chain as L2 . Szabo, Schul-ten, and Schulten, (J. Chem. Phys. 72, 4350 (1980)) have used mean first passage time approach and they find that the closure time vary as L3/2. Both approaches have been compared with simulations (Pastor et.al, J. Chem. Phys., 105, 3878 (1996), Srinivas et. al,116,7276 (2002)). In the case of semiflexible chains, studies are fewer in comparison. However, real polymers such as DNA, RNA or proteins are not flexible and therefore, it is important to incorporate the intrinsic stiffness of the chain into account. In the worm-like chain model, the chain is described as a continuous, inextensible and differentiable space curve represented by the position vector r(s), where s is the arc length. Inextensibility of the chain requires that the tangent vector, u(s) = ∂r(s)/∂s, at any point on the curve must have unit magnitude, i.e, |u(s)| = 1. But incorporating this constraint has been a difficult problem in dealing with semiflexible polymers. Yamakawa and Stockmayer (J. Chem. Phys., 57, 2843 (1972)) and Shimada and Yamakawa (Macromolecules, 17, 689 (1984)) have calculated ring closure probabilities for worm-like chains and helical worm-like chains. Cherayil and Dua (J. Chem. Phys., 116, 399 (2002)) have calculated closure time for a semiflexible chain using the approximate model for semiflexible chains by Har-nau, Winkler and Reineker (J. Chem. Phys., 101, 8119 (1994)) and find that the closure time ~ Lν where ν is in the range 2.2 to 2.4. Physically, one expects that the closing time should decrease exponentially with length in the very short chain limit and then increase with length for longer chains. Hence, the closing time has a minimum at an intermediate length. The reason for this behavior is that, for short chains, the bending energy contributes significantly to the activation energy for the process. The activation energy ~ const./L and hence, the closing time τ ~ exp(const./L). For longer chains, the free energy barrier for closing is due to the configurational entropy and hence, τ obeys a power law. Recently, Jun et. al (Europhys. Lett., 64, 420 (2003)) have followed an approximate one dimensional Kramers approach to reproduce this behavior and obtained the minimum at a length Lmin = 3.4lp, where lp is the persistence length of the chain. Monte Carlo simulations by Chen et.al (Europhys. Lett., 65, 407 (2004)) lead Lmin = 2.85lp. We investigate (K. P. Santo and K. L. Sebastian, Phys. Rev. E, 73, 031923, (2006)) in detail the problem of loop opening for semiflexible polymers. The inextensibility constraint is incorporated rigorously by setting u(s) to be a unit vector in the angular direction (θ, φ) and the conformations of the polymer are then represented by Brownian motion over a unit sphere in the tangent vector space. We use the worm-like chain model, which takes into account the bending rigidity of the polymer. The bending energy can then be given in terms of the angle coordinates θ and φ. For the dynamics, we make use of a semiclassical approach, which is based on expanding the bending energy about a minimum energy path. For the sake of simplicity, we take the great circle on the unit sphere to be the minimum energy configuration of the loop and expand the bending energy up to second order in terms of fluctuations about this configuration. We find that, this is a very good approximation in the large stiffness limit, as this approach leads to a minimum energy value, which is very close to the exact calculations. The loop is unstable, unless the ends are bound to each other with a potential. Once the two ends have been brought together, they can separate from each other in any of the three directions in space. Considering the ring to be in the XY plane with the ends meeting in the Y-axis, we find that the separation in the X and Z directions are unstable as motion in these directions lead to decrease in bending energy. But the motion in the other direction, that is, the Y direction leads to increase in energy and is stable. Therefore, we choose the potential to be of Morse type in the X-direction and stable harmonic ones in the other two directions. With this, the potential energy surface for opening can be found and the rate of opening can be calculated using classical Transition State Theory. The effects of friction on the rate can also be incorporated using the standard coupling to a bath of harmonic oscillators . We find that for short chains, the rate is strongly length dependent and is well-described by the equation Aexp(B/x)/xν, with A and B constants, x = L/lp, L the length of the chain, lp the persistence length and ν ~ 1.2. However, for long chains, the rate is found to obey a power law. But in view of the fact that our approximations, while sensible for short semiflexible chains, are not expected to be valid for long flexible chains and therefore, this result is not expected to be correct. We also present results for the seemingly more biologically important reverse process, the closing of a semiflexible polymer, thus presenting a rather complete theory of dynamics of semiflexible polymer loops. In this work, we give a detailed multidimensional analysis of the closing dynamics of semiflexible chains by making use of the approximation scheme developed in the previous study of loop opening. We use the formalism of Wilemski and Fixman for the diffusion-controlled intra-chain reactions of polymers and their "closure" approximation for an arbitrary sink function. In this procedure, the closing time is expressed in terms of a sink-sink correlation function. We calculate this sink-sink correlation function through a normal mode analysis on the chain. The closing times, τclose for different lengths of the chain are then obtained. We find that τclose(L) ~ L4.5W(L), where W(L) was found to be described by B'exp(A'/L) with A' and B' constants. τclose(L) is found to have a minimum at Lmin = 2.4lp, which is to be compared with the values obtained through a one dimensional analysis (Europhys. Lett., 64, 420 (2003)) and simulations (Europhys. Lett., 65, 407 (2004)). We thus present a multidimensional analysis that give results that are physically expected. There does not seem to be any previous analysis which leads to these results shown through one-dimensional studies and simulations. In the category of translocation problems, we consider DNA packaging in viruses. DNA Packaging into the viral capsid is an essential step in any kind of viral infection. The mechanism of packaging in bacteriophage φ — 29 has recently been studied (Simpson et. al, Nature (London), 408, 745 (2000)). The study revealed the structure of the molecular motor that packages the DNA. In another experimental study, Smith et. Al (Nature (London), 408, 745 (2001)) have investigated the effect of applied external force on the packaging. Motivated by this study, we suggest (K. P. Santo and K. L. Sebastian, Phys. Rev. E, 65, 052902 (2002)) a simple model to explain the kinetics of packaging of DNA the external force, which tries to prevent it. The model suggests a Butler-Volmer kind of dependence of the rate of packaging on the pulling force. We find that our model explains the experimental data very well. Another very interesting situation that arises in biological contexts is the translocation of a polymer across a membrane through a pore. The uptake of DNA into the cell nucleus and the translocation of cytosolic protein into endoplasmic reticulum are examples. There have been two main classes of polymer translocation problems; translocation in presence of a field or driven by a molecular motor and the translocation assisted by the adsorption of molecules onto the chain in the region into which it is translocated. While the first class of problems is reasonably well understood, for the second class of problems a complete understanding still does not exist in the literature. The existing understanding of this kind of polymer translocation is mainly due to Simon, Peskin and Oster (Proc. Natl. Accad. Sci. USA, 89, 3770 (1992)), who describe the translocation as kind of biased Brownian motion, which is known as the Brownian Ratchet. But Brownian Ratchet is an idealization and can only be realized in certain limits and therefore, it does not account for the detailed dynamics of polymer and the binding particles. We present a simple statistical description of the problem. We find that in the regime where number of binding particles are larger than the number of adsorption sites on the chain, the translocation proceeds as if it is driven by a constant force and hence, seems to be governed by a mechanism similar to the kink mechanism (K. L. Sebastian and Alok. K. R. Paul, Phys. Rev. E, 62, 927 (2000), K. L. Sebastian, 61, 3245 (2000)) that has been suggested in the case translocation in presence of an external field. In the other regime, where the number of binding particles are less than the number of binding sites on the chain, the translocation was found to be predominantly diffusive.

Die Bildung infektiöser Viruspartikel des humanen Zytomegalievirus (HCMV) ist ein mehr-stufiger Prozess. Sie beginnt mit der Verpackung der DNA in die Kapside im Kern, gefolgt von weiterer Reifung während des Transports durch das Zytoplasma und der abschließenden Freisetzung aus der Zelle. Im Zuge dieser Arbeit wurden zwei Proteine, die Einfluss auf die ebengenannten Prozesse haben, analysiert. Der erste Teil der Arbeit befasst sich mit der funktionellen Charakterisierung des HCMV Pro-teins pUL77. Es ist bekannt, dass das homologe Protein pUL25 in alpha-Herpesvirinae essentiell für die DNA-Verpackung ist. Zunächst konnte das Protein als Kapsid-assoziiertes strukturelles Protein identifiziert werden. Es wurden Interaktionen von pUL77 mit DNA-Verpackungs- und Kapsidproteinen gezeigt. Weiterhin wurde die DNA-Bindungsfähigkeit von pUL77 in verschiedenen „in vitro“-Experimenten untersucht. Zusammengefasst weisen unsere Ergebnisse auf eine Funktion von HCMV pUL77 bei der DNA-Verpackung hin. Im zweiten Teil der Arbeit wurde das HCMV Protein pUL71 charakterisiert, das in allen Herpesviren konserviert vorkommt, dessen Funktion jedoch nicht charakterisiert ist. Zunächst wurde das Protein als strukturelles Tegumentprotein mit “earlylate“ Expressionskinetik klassifiziert. Weiterhin wurden die subzelluläre Lokalisation sowie virale und zelluläre Interaktionspartner untersucht. Die Ergebnisse weisen auf eine Funktion von HCMV pUL71 bei der Reifung und beim Transport der Virionen im Zytoplasma hin. „In silico“-Vorhersagen zeigten ein „Leuzin Zipper“-Motiv in pUL71, das als mögliche Oligomerisationsdomäne dienen könnte. Mutationen wurden in dieses Motiv eingebracht und die resultierenden Proteine auf ihre Oligomerisationsfähigkeit mit „in vitro“-Methoden und in rekombinanten Viren untersucht. Zusammenfassend konnten wir zeigen, dass das „Leuzin Zipper“-Motiv wichtig für die Funktion von pUL71 ist und diese mit einer unbeeinträchtigten Oligomerisation des Proteins zusammen hängt. / The morphogenesis of Human cytomegalovirus (HCMV) virions starts with the capsid assem-bly and DNA insertion in the nucleus followed by maturation during transport through the cytoplasm prior to release of virus progeny. In this study we are functionally characterising two proteins that are involved in those steps. The function of essential HCMV protein pUL77 is characterised in the first part of the study. HCMV pUL77 was shown to be a structural protein associated with capsids. Furthermore, our experiments demonstrated that HCMV pUL77 interacts with DNA packaging motor compo-nents and capsid proteins. The ability of HCMV pUL77 to bind double-stranded DNA was studied in “in vitro” assays designed for this study. The homologue α-Herpesvirinae protein pUL25 is described to be involved in processes connected with DNA packaging. Data ob-tained in this study demonstrates that HCMV pUL77 might serve a similar function. In the second part of the study HCMV pUL71, conserved throughout the Herpesvirus family but to date unclassified, was functionally characterised. HCMV pUL71 was defined a struc-tural tegument protein with early-late expression kinetics. We studied the sub-cellular local-isation and interactions of pUL71 with a subset of cellular and viral proteins. Thereby we could show that HCMV pUL71 function might be connected with processes of viral egress. By in silico analyses we identified a leucine zipper motif in pUL71 that might serve as a puta-tive oligomerisation domain. In order to investigate the function of the leucine zipper motif, we performed in vitro assays and investigated the alterations of the motif in the viral context. Taken together we can conclude that (i) an intact leucine zipper motif is crucial for the func-tion of pUL71 and (ii) this function is dependent upon undisturbed oligomerisation of the pro-tein.

Die Verpackung und Spaltung konkatemerer DNA ist ein essentieller Prozeß bei der Reifung von Virionen. Die an diesem Prozess maßgeblich beteiligten Proteine werden als Terminase bezeichnet. Die Inhibition der HCMV Terminase bietet einen attraktiven alternativen Ansatzpunkt für die antivirale Therapie. Zur Inhibition der Terminase Aktivität wurden die neuen Benzimidazol D-Ribonukleosid Derivate BTCRB und Cl4RB auf ihre antivirale Wirkung analysiert. Die neuen Benzimidazol D-Ribonukleosid Derivate BTCRB und Cl4RB zeigten sowohl gegen den HCMV Laborstamm AD169 als auch gegen klinische HCMV-Isolaten eine Wirkung. Weiterhin wurde die Wirksamkeit der Substanzen auf anderen Herpesviren getestet. Interessanterweise zeigten BTCRB und Cl4RB sowohl einen Effekt gegen Varicella-Zoster-Virus (VZV) als auch Ratten-Cytomegalovirus (RCMV), wohingegen der Effekt gegen Herpes-simplex-Virus Typ-1 (HSV-1) und Maus-Cytomegalovirus (MCMV) gering war. Infolgedessen eignen sich beide Substanzen BTCRB und Cl4RB als attraktive alternative Inhibitoren für die weitere Entwicklung einer antiviralen Therapie. / DNA packaging is the key step in viral maturation and involves binding and cleavage of viral DNA containing specific DNA-packaging motifs. This process is mediated by a group of specific enzymes called terminase. The development for an inhibitor of HCMV terminase would be of great value, because it would act subsequent to DNA synthesis and block the first steps in viral maturation. Therefore we characterized two inhibitors targeting the HCMV terminase, 2-bromo-4,5,6-trichloro-1-(2,3,5-tri-O-acetly-ß-D-ribofuranosyl) benzimidazole (BTCRB) and 2,4,5,6-tetrachloro-1-(2,3,5-tri-O-acetly-ß-D-ribofuranosyl) benzimidazole (Cl4RB). By using viral plaque formation, viral yield, viral growth kinetics and electron microscopy, we demonstrated that two compounds BTCRB und Cl4RB are highly active against HCMV AD 169 and HCMV clinical isolates. In addition, the antiviral effect on other herpesviruses was determined. Interestingly BTCRB was active all tested herpesviruses. The best effects were observed on VZV-and RCMV-infected cells. Therefore new compounds might be promising attractive compounds for antiviral therapy in the future.

Der DNA-Verpackungsmechanismus des humanen Cytomegalievirus (HCMV) ist charakteristisch für große DNA-Viren wie Herpesviren und ds-Bakteriophagen. Er beruht auf der Spaltung der konkatemeren DNA durch einen viralen, hetero-oligomeren Proteinkomplex, der Terminase. In der vorliegenden Arbeit konnten die funktionellen Domänen der Terminase-Untereinheit pUL89 in vitro identifiziert und charakterisiert werden. Neben einer Nuklease-Aktivität besitzt pUL89 auch die Fähigkeit dsDNA sequenz-unabhängig zu binden. Durch Nuklease-Untersuchungen konnte gezeigt werden, dass pUL89 sowohl dsDNA, als auch lineare DNA spaltet. PUL89 weist dabei eine größere Spezifität zu dsDNA auf. Des Weiteren konnte nachgewiesen werden, dass die Aminosäure D463 eine zentrale Funktion innerhalb der Nuklease-Aktivität besitzt. Durch kolorimetrische DNA-Bindungsuntersuchungen konnte die Aminosäure R544 als essenziell für die dsDNA-Bindungsfähigkeit von pUL89 identifiziert werden. Basierend auf den in vitro Ergebnissen wurden rekombinanten TB40/E-Virusmutanten mit Mutationen im ORF UL89 durch die En Passant Mutagenese generiert. Mit Hilfe dieser Viren sollte der Einfluss der Mutationen auf die Replikation des Virus charakterisiert werden. Es war möglich nachzuweisen, dass die Aminosäuren E534 und R544 eine essenzielle Aufgabe innerhalb von HCMV erfüllen, da die Mutation einer dieser Aminosäure zu nicht wachstums-fähigen BAC-Mutanten führte. Zur Charakterisierung dieser Konstrukte wurden die Zelllinien HELF Fi301-UL89 und HELF Fi301-vProm-UL89 verwendet. Durch Untersuchungen hinsichtlich der Wachstumseigenschaften, Proteinexpression, DNA-Spaltung, DNA-Bindung sowie elektronenmikroskopischen Aufnahmen, konnte gezeigt werden, dass die wachstums-kompetenten BAC-Mutanten keinen signifikanten Unterschied zum Wildtyp-Virus TB40/E zeigten. Sodass nachgewiesen werden konnte, dass die basischen Aminosäuren H565 und H571 keine essenzielle Funktion in pUL89 erfüllen. / The human cytomegalovirus DNA packaging mechanism is characteristic for large DNA viruses like Herpes viruses and ds bacteriophages. This mechanism is based on the cleavage of concatemeric DNA by the viral heterooligomeric protein complex terminase. This dissertation includes the identification and characterization of functional domains of the HCMV terminase subunit pUL89. PUL89 contain a nuclease activity and the ability to bind dsDNA. This protein shows the property to cut as well dsDNA as linear DNA. The amino acid D463 shows a significant role in this cleavage event. Colorimetric DNA binding experiments show the central role of R544 in DNA binding by pUL89. Based of the in vitro results recombinant TB40/E viruses with mutations in the ORF UL89 were generated. These viruses allow a characterization of the impact of virus replication. It was possible to show that the amino acids E534 and R544 have a functional role in HCMV. The mutation of one of these amino acids was enough to generate a growth deficient mutant. The stable cell lines HELF Fi301-UL89 and HELF Fi301-vProm UL89 were used for the characterization of the growth deficient mutants. The growth competent mutants H656A and H571A show no significant differences in comparison with the wild type TB40/E virus. This was verified by growth kinetics, protein expression characterizations, pulse field gel electrophoresis, DNA binding assays and electron microscopy.

Many double-stranded DNA viruses use a packaging motor during maturation to recognize and transport genetic material into the capsid. In terminase motors, the TerS complex recognizes DNA, while the TerL motor packages the DNA into the capsid shell. Although there are several models for DNA recognition and translocation, how the motor components assemble and power DNA translocation is unknown. Using the thermophilic P74-26 bacteriophage model system, we discover that TerL uses a trans-activated ATP hydrolysis mechanism. Additionally, we identify critical residues for TerL ATP hydrolysis and DNA binding. With a combination of x-ray crystallography, SAXS, and molecular docking, we build a structural model for TerL pentamer assembly. Apo and ATP analog-bound TerL ATPase domain crystal structures show ligand-dependent conformational changes, which we propose power DNA translocation. Together, we assimilate these findings to build models for both motor assembly and DNA translocation. Additionally, with the P76-26 system, we identify the TerS protein as gp83. I find that P74-26 TerS is a nonameric ring that stimulates TerL ATPase activity while inhibiting TerL nuclease activity. Using cryoEM, I solve 3.8 Å and 4.8 Å resolution symmetric and asymmetric reconstructions of the TerS ring. I observe in P74-26 TerS, the conserved C-terminal beta-barrel is absent, and instead the region is flexible or unstructured. Furthermore, the helix-turn-helix motifs of P74-26 TerS are positioned differently than those of known TerS structures, suggesting P74-26 uses an alternative mechanism to recognize DNA.