Mutational analysis of active-site residues in the Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease: Asp(122) and Asp(193) are crucial to the double-stranded DNA cleavage activity whereas Asp(218) is not

Mycobacterium leprae recA harbors an in-frame insertion sequence that encodes an intein homing endonuclease (PI-MleI). Most inteins (intein endonucleases) possess two conserved LAGLIDADG (DOD) motifs at their ctive center. A common feature of LAGLIDADG-type homing endonucleases is that they recognize and cleave the same or very similar DNA sequences. However, PI-MleI is distinctive from other members of the family of LAGLIDADG-type HEases for its modular structure with functionally separable domains for DNA-binding and cleavage, each with distinct sequence preferences. Sequence alignment analyses of PI-MleI revealed three putative LAGLIDADG motifs; however, there is conflicting bioinformatics data in regard to their identity and specific location within the intein polypeptide. To resolve this conflict and to determine the active-site residues essential for DNA target site recognition and double-stranded DNA cleavage, we performed site-directed mutagenesis of presumptive catalytic residues in the LAGLIDADG motifs. Analysis of target DNA recognition and kinetic parameters of the wild-type PI-MleI and its variants disclosed that the two amino acid residues, Asp(122) (in Block C) and Asp(193) (in functional Block E), are crucial to the double-stranded DNA endonuclease activity, whereas Asp(218) (in pseudo-Block E) is not. However, despite the reduced catalytic activity, the PI-MleI variants, like the wild-type PI-MleI, generated a footprint of the same length around the insertion site. The D122T variant showed significantly reduced catalytic activity, and D122A and D193A mutations although failed to affect their DNA-binding affinities, but abolished the double-stranded DNA cleavage activity. On the other hand, D122C variant showed approximately twofold higher double-stranded DNA cleavage activity, compared with the wild-type PI-MleI. These results provide compelling evidence that Asp(122) and Asp(193) in DOD motif I and II, respectively, are bona fide active-site residues essential for DNA cleavage activity. The implications of these results are discussed in this report.

RecA Protein of Mycobacterium tuberculosis Possesses pH-Dependent Homologous DNA Pairing and Strand Exchange Activities: Implications for Allele Exchange in Mycobacteria

To gain insights into inefficient allele exchange in mycobacteria, we compared homologous pairing and strand exchange reactions promoted by RecA protein of Mycobacterium tuberculosis to those of Escherichia coli RecA protein. The extent of single-stranded binding protein (SSB)-stimulated formation of joint molecules by MtRecA was similar to that of EcRecA over a wide range of pH values. In contrast, strand exchange promoted by MtRecA was inhibited around neutral pH due to the formation of DNA networks. At higher pH, MtRecA was able to overcome this constraint and, consequently, displayed optimal strand exchange activity. Order of addition experiments suggested that SSB, when added after MtRecA, was vital for strand exchange. Significantly, with shorter duplex DNA, MtRecA promoted efficient strand exchange without network formation in a pH-independent fashion. Increase in the length of duplex DNA led to incomplete strand exchange with concomitant rise in the formation of intermediates and networks in a pH-dependent manner. Treatment of purified networks with S1 nuclease liberated linear duplex DNA and products, consistent with a model in which the networks are formed by the invasion of hybrid DNA by the displaced linear single-stranded DNA. Titration of strand exchange reactions with ATP or salt distinguished a condition under which the formation of networks was blocked, but strand exchange was not significantly affected. We discuss how these results relate to inefficient allele exchange in mycobacteria.

Antibodies raised against denatured DNA bind to double-stranded DNA

Antibodies raised against denatured DNA complexed with methylated bovine serum albumin have been reported to react with ssDNA but not with dsDNA. Using a highly sensitive avidin-biotin microELISA, we report that such antibodies also bind to dsDNA. Antibodies which reacted with ssDNA and dsDNA were found to be IgG type. The antibodies did not react with tRNA and rRNA. The binding of antibodies to dsDNA was partially inhibited dy individual deoxyribonucleotides. ssDNA as well as dsDNA inhibited the binding of antibodies to dsDNA. The binding of these antibodies to supercoiled and relaxed forms of pBR322 DNA was demonstrated by gel retardation assay. The cross-reaction with ssDNA was observed even after affinity purification on native DNA-cellulose. The antibodies were also shown to bind to poly(dA-dT)·poly(dA-dT)

Optical Tweezers for Single Molecule Biology

Molecular machinery on the micro-scale, believed to be the fundamental building blocks of life, involve forces of 1-100 pN and movements of nanometers to micrometers. Micromechanical single-molecule experiments seek to understand the physics of nucleic acids, molecular motors, and other biological systems through direct measurement of forces and displacements. Optical tweezers are a popular choice among several complementary techniques for sensitive force-spectroscopy in the field of single molecule biology. The main objective of this thesis was to design and construct an optical tweezers instrument capable of investigating the physics of molecular motors and mechanisms of protein/nucleic-acid interactions on the single-molecule level. A double-trap optical tweezers instrument incorporating acousto-optic trap-steering, two independent detection channels, and a real-time digital controller was built. A numerical simulation and a theoretical study was performed to assess the signal-to-noise ratio in a constant-force molecular motor stepping experiment. Real-time feedback control of optical tweezers was explored in three studies. Position-clamping was implemented and compared to theoretical models using both proportional and predictive control. A force-clamp was implemented and tested with a DNA-tether in presence of the enzyme lambda exonuclease. The results of the study indicate that the presented models describing signal-to-noise ratio in constant-force experiments and feedback control experiments in optical tweezers agree well with experimental data. The effective trap stiffness can be increased by an order of magnitude using the presented position-clamping method. The force-clamp can be used for constant-force experiments, and the results from a proof-of-principle experiment, in which the enzyme lambda exonuclease converts double-stranded DNA to single-stranded DNA, agree with previous research. The main objective of the thesis was thus achieved. The developed instrument and presented results on feedback control serve as a stepping stone for future contributions to the growing field of single molecule biology.

Nucleosomes on linear duplex DNA allow homologous pairing but prevent strand exchange promoted by RecA protein

To understand the molecular basis of gene targeting, we have studied interactions of nucleoprotein filaments comprised of single-stranded DNA and RecA protein with chromatin templates reconstituted from linear duplex DNA and histones. We observed that for the chromatin templates with histone/DNA mass ratios of 0.8 and 1.6, the efficiency of homologous pairing was indistinguishable from that of naked duplex DNA but strand exchange was repressed. In contrast, the chromatin templates with a histone/DNA mass ratio of 9.0 supported neither homologous pairing nor strand exchange. The addition of histone H1, in stoichiometric amounts, to chromatin templates quells homologous pairing. The pairing of chromatin templates with nucleoprotein filaments of RecA protein-single-stranded DNA proceeded without the production of detectable networks of DNA, suggesting that coaggregates are unlikely to be the intermediates in homologous pairing. The application of these observations to strategies for gene targeting and their implications for models of genetic recombination are discussed.

DNA Binding, Coprotease, and Strand Exchange Activities of Mycobacterial RecA Proteins: Implications for Functional Diversity among RecA Nucleoprotein Filaments

One of the fundamental questions concerning homologous recombination is how RecA or its homologues recognize several DNA sequences with high affinity and catalyze all the diverse biological activities. In this study, we show that the extent of single-stranded DNA binding and strand exchange (SE) promoted by mycobacterial RecA proteins with DNA substrates having various degrees of GC content was comparable with that observed for Escherichia coli RecA. However, the rate and extent of SE promoted by these recombinases showed a strong negative correlation with increasing amounts of sequence divergence embedded at random across the length of the donor strand. Conversely, a positive correlation was seen between SE efficiency and the degree of sequence divergence in the recipient duplex DNA. The extent of heteroduplex formation was not significantly affected when both the pairing partners contained various degrees of sequence divergence, although there was a moderate decrease in the case of mycobacterial RecA proteins with substrates containing larger amounts of sequence divergence. Whereas a high GC content had no discernible effect on E. coli RecA coprotease activity, a negative correlation was apparent between mycobacterial RecA proteins and GC content. We further show clear differences in the extent of SE promoted by E. coli and mycobacterial RecA proteins in the presence of a wide range of ATP:ADP ratios. Taken together, our findings disclose the existence of functional diversity among E. coli and mycobacterial RecA nucleoprotein filaments, and the milieu of sequence divergence (i.e., in the donor or recipient) exerts differential effects on heteroduplex formation, which has implications for the emergence of new genetic variants.

DNA polymerase alpha-primase complex from the silk glands of the non-mulberry silkworm Philosamia ricini.

The DNA content in the silk glands of the non-mulberry silkworm Philosamia ricini increases continuously during the fourth and fifth instars of larval development indicating high levels of DNA replication in this terminally differentiated tissue. Concomitantly, the DNA polymerase alpha activity also increases in the middle and the posterior silk glands during development, reaching maximal levels in the middle of the fifth larval instar. A comparable level of DNA polymerase delta/epsilon was also observed in this highly replicative tissue. The DNA polymerase alpha-primase complex from the silk glands of P. ricini has been purified to homogeneity by conventional column chromatography as well as by immunoaffinity techniques. The molecular mass of the native enzyme is 560 kDa and the enzyme comprises six non-identical subunits. The identity of the enzyme as DNA polymerase alpha has been established by its sensitivity to inhibitors such as aphidicolin, N-ethylmaleimide, butylphenyl-dGTP, butylanilino-dATP and antibodies to polymerase alpha. The enzyme possesses primase activity capable of initiating DNA synthesis on single-stranded DNA templates. The tight association of polymerase and primase activities at a constant ratio of 6:1 is observed through all the purification steps. The 180 kDa subunit harbours the polymerase activity, while the primase activity is associated with the 45 kDa subunit.

Excision of uracil from the ends of double stranded DNA by uracil DNA glycosylase and its use in high efficiency cloning of PCR products

We show that uracil DNA glycosylase from E. coli excises uracil residues from the ends of double stranded oligos. This information has allowed us to develop an efficient method of cloning PCR amplified DNA. In this report, we describe use of this method in cloning of E. coli genes for lysyl- and methionyl-tRNA synthetases. Efficiency of cloning by this method was found to be the same as that of subcloning of DNA restriction fragments from one vector to the other vector. Possibilities of using other DNA glycosylases for such applications are discussed.

Unwinding of heterologous DNA by RecA protein during the search for homologous sequences

The search for homologous sequences promoted by RecA protein in vitro involves a presynaptic filament and naked duplex DNA, the multiple contacts of which produce nucleoprotein networks or coaggregates. The single-stranded DNA within the presynaptic filaments, however, is extended to an axial spacing 1.5 times that of B-form DNA. To investigate this paradoxical difference between the spacing of bases in the RecA presynaptic filament versus the target duplex DNA, we explored the effect of heterologous contacts on the conformation of DNA, and vice versa. In the presence of wheat germ topoisomerase I, RecA presynaptic filaments induced a rapid, limited reduction in the linking number of heterologous circular duplex DNA. This limited unwinding of heterologous duplex DNA, termed heterologous unwinding, was detected within 30 seconds and reached a steady state within a few minutes. Presynaptic filaments that were formed in the presence of ATP?S and separated from free RecA protein by gel filtration also generated a ladder of topoisomers upon incubation with relaxed duplex DNA and topoisomerase. The inhibition of heterologous contacts by 60 mImage -NaCl or 5 mImage -ADP resulted in a corresponding decrease in heterologous unwinding. In reciprocal fashion, the stability or number of heterologous contacts with presynaptic filaments was inversely related to the linking number of circular duplex DNA. These observations show that heterologous contacts with the presynaptic filament cause a limited unwinding of the duplex DNA, and conversely that the ability of the DNA to unwind stabilizes transient heterologous contacts.

DNA topoisomerase I from mycobacteria - A potential drug target

DNA topoisomerases are ubiquitous group of enzymes altering the topology of DNA by concerted breakage and rejoining of the phosphodiester backbone of DNA. The enzymes are classified based on the pattern of DNA cleavage. Type IA enzymes found in all bacteria nick the DNA and attach themselves covalently to the 5' side of the nick during the first transesterification reaction. Most of the information on this group of enzymes comes from studies with E. coli topoisomerase I and III. Members of type IA group are single subunit Zn++ metalloenzymes recognizing single stranded DNA without high degree of sequence specificity during relaxation reaction of negatively super coiled DNA. So far no inhibitors are known for this group of enzymes inspite of their important role in maintaining homeostasis of DNA topology. Molecular characterization of DNA topoisomerase I from mycobacteria has revealed some of the important features of type IA enzymes hitherto unknown and provide scope for identifying novel inhibitors. The present review describes the recent developments in the area summarizing the distinctive features of mycobacterial topoisomerase I. The enzyme has several properties not shared by either type IA or 113 enzymes with respect to DNA binding, recognition, sequence specificity and interaction pattern. The physiological basis of the unusual features is discussed. The unique properties described would aid in developing the enzyme as a target molecule in pharmaceutical design. In addition, the findings lead to address some fundamental questions on the intracellular role of topoisomerase I in the biology of mycobacteria which are one of the most formidable group of pathogenic organisms.

Helicobacter pylori DprA alleviates restriction barrier for incoming DNA

Helicobacter pylori is a Gram-negative bacterium that colonizes human stomach and causes gastric inflammation. The species is naturally competent and displays remarkable diversity. The presence of a large number of restriction-modification (R-M) systems in this bacterium creates a barrier against natural transformation by foreign DNA. Yet, mechanisms that protect incoming double-stranded DNA (dsDNA) from restriction enzymes are not well understood. A DNA-binding protein, DNA Processing Protein A (DprA) has been shown to facilitate natural transformation of several Gram-positive and Gram-negative bacteria by protecting incoming single-stranded DNA (ssDNA) and promoting RecA loading on it. However, in this study, we report that H. pylori DprA (HpDprA) binds not only ssDNA but also dsDNA thereby conferring protection to both from various exo-nucleases and Type II restriction enzymes. Here, we observed a stimulatory role of HpDprA in DNA methylation through physical interaction with methyltransferases. Thus, HpDprA displayed dual functional interaction with H. pylori R-M systems by not only inhibiting the restriction enzymes but also stimulating methyltransferases. These results indicate that HpDprA could be one of the factors that modulate the R-M barrier during inter-strain natural transformation in H. pylori.

N-Terminal disordered domain of saccharomyces cerevisiae Hop1 protein Is dispensable for DNA binding, bridging, and synapsis of double-stranded DNA molecules but is ecessary for spore formation

The cytological architecture of the synaptonemal complex (SC), a meiosis-specific proteinaceous structure, is evolutionarily conserved among eukaryotes. However, little is known about the biochemical properties of SC components or the mechanisms underlying their roles in meiotic chromosome synapsis and recombination. Functional analysis of Saccharomyces cerevisiae Hop1, a key structural component of SC, has begun to reveal important insights into its function in interhomolog recombination. Previously, we showed that Hop1 is a structure-specific DNA-binding protein, exhibits higher binding affinity for the Holliday junction, and induces structural distortion at the core of the junction. Furthermore, Hop1 promotes DNA condensation and intra- and intermolecular synapsis between duplex DNA molecules. Here, we show that Hop1 possesses a modular domain organization, consisting of an intrinsically disordered N-terminal domain and a protease-resistant C-terminal domain (Hop1CTD). Furthermore, we found that Hop1CTD exhibits strong homotypic as well as heterotypic protein protein interactions, and its biochemical activities were similar to those of the full-length Hop1 protein. However, Hop1CTD failed to complement the meiotic recombination defects of the Delta hop1 strain, indicating that both N- and C-terminal domains of Hop1 are essential for meiosis and spore formation. Altogether, our findings reveal novel insights into the structure-function relationships of Hop1 and help to further our understanding of its role in meiotic chromosome synapsis and recombination.

Detection of Hepatitis B DNA Sequences on Polyelectrolyte Based Non-Covalently Functionalized Flexible Plastic Substrates

Development of simple functionalization methods to attach biomolecules such as proteins and DNA on inexpensive substrates is important for widespread use of low cost, disposable biosensors. Here, we describe a method based on polyelectrolyte multilayers to attach single stranded DNA molecules to conventional glass slides as well as a completely non-standard substrate, namely flexible plastic transparency sheets. We then use the functionalized transparency sheets to specifically detect single stranded Hepatitis B DNA sequences from samples. We also demonstrate a blocking method for reducing non-specific binding of target DNA sequences using negatively charged polyelectrolyte molecules. The polyelectrolyte based functionalization method, which relies on surface charge as opposed to covalent surface linkages, could be an attractive platform to develop assays on inexpensive substrates for low cost biosensing.

Detection of Target DNA using Photo-Reactive Protoporphyrin Moeity on a Nanocomposite Substrate

Detection of pathogens from infected biological samples through conventional process involves cell lysis and purification. The main objective of this work is to minimize the time and sample loss, as well as to increase the efficiency of detection of biomolecules. Electrical lysis of medical sample is performed in a closed microfluidic channel in a single integrated platform where the downstream analysis of the sample is possible. The device functions involve, in a sequence, flow of lysate from lysis chamber passed through a thermal denaturation counter where dsDNA is denatured to ssDNA, which is controlled by heater unit. A functionalized binding chamber of ssDNA is prepared by using ZnO nanorods as the matrix and functionalized with bifunctional carboxylic acid, 16-(2-pyridyldithiol) hexadecanoic acid (PDHA) which is further attached to a linker molecule 1-ethyl-3-(3-dimethylaminopropyl) (EDC). Linker moeity is then covalently bound to photoreactive protoporphyrin (PPP) molecule. The photolabile molecule protoporphyrin interacts with -NH2 labeled single stranded DNA (ssDNA) which thus acts as a probe to detect complimentary ssDNA from target organisms. Thereafter the bound DNA with protoporphyrin is exposed to an LED of particular wavelength for a definite period of time and DNA was eluted and analyzed. UV/Vis spectroscopic analysis at 260/280 nm wavelength confirms the purity and peak at 260 nm is reconfirmed for the elution of target DNA. Quantitative and qualitative data obtained from the current experiments show highly selective detection of biomolecule such as DNA which have large number of future applications in Point-of-Care devices.

DNA Assisted Self-Assembly of PAMAM Dendrimers

We report DNA assisted self-assembly of polyamidoamine (PAMAM) dendrimers using all atom Molecular Dynamics (MD) simulations and present a molecular level picture of a DNA-linked PAMAM dendrimer nanocluster, which was first experimentally reported by Choi et al. (Nano Lett., 2004, 4, 391-397). We have used single stranded DNA (ssDNA) to direct the self-assembly process. To explore the effect of pH on this mechanism, we have used both the protonated (low pH) and nonprotonated (high pH) dendrimers. In all cases studied here, we observe that the DNA strand on one dendrimer unit drives self-assembly as it binds to the complementary DNA strand present on the other dendrimer unit, leading to the formation of a DNA-linked dendrimer dimeric complex. However, this binding process strongly depends on the charge of the dendrimer and length of the ssDNA. We observe that the complex with a nonprotonated dendrimer can maintain a DNA length dependent inter-dendrimer distance. In contrast, for complexes with a protonated dendrimer, the inter-dendrimer distance is independent of the DNA length. We attribute this observation to the electrostatic complexation of a negatively charged DNA strand with the positively charged protonated dendrimer.