Tag: Andrew Marcus

Single molecule polarization sweep spectroscopy

Presenter: Anabel Chang − Biochemistry

Faculty Mentor(s): Andrew Marcus, Jack Maurer

(In-Person) Poster Presentation

Local fluctuations of the sugar-phosphate backbones of DNA (a form of DNA ‘breathing’) play key roles in protein-DNA assembly and enzymatic function. By monitoring spectroscopic signals from single-molecules of DNA constructs labeled with optical probes rigidly inserted within the sugar- phosphate backbones at opposite positions within complementary single-strands, it is possible to study local conformational fluctuations within DNA at specific sites. Here we present an experimental single-molecule spectroscopic method, to monitor the local fluctuations of Cy3-labeled DNA constructs at varying positions near a single-stranded (ss)-double-stranded (ds)DNA junction. The method combines single-molecule total internal reflection fluorescence (TIRF) microscopy with polarized, phase-modulated optical excitation to detect linear optical signals. We use a linearly polarized continuous wave (cw) laser beam to excite the single-molecule sample, so that the emitted fluorescence contains information about the relative conformational changes of an exciton-coupled Cy3 dimer probe that labels the DNA sugar-phosphate backbones. Our results indicate that the local conformation of DNA at positions near ss-dsDNA junctions adopts four topologically-relevant macrostates. We apply a kinetic network model approach to interpret our observations of DNA breathing fluctuations at and near the ss-dsDNA junction.

Investigating Gp32 Binding Behavior on Single-Stranded DNA With Different Polarity And Length Using Microsecond Resolution smFRET Measurements

Presenter(s): Anson Dang

Faculty Mentor(s): Andrew Marcus & Peter von Hippel

Poster 67

Session: Sciences

The single-stranded (ss)DNA binding protein (gp32) of bacteriophage T4 plays a central role in regulating the functions and integration of the helicase, polymerase and primase components of the T4 DNA replication system. The T4 replication system serves as an excellent model for higher organisms as it contains all the essential components for DNA replication. This project aims to investigate how polarity and length of the ssDNA affect gp32 DNA binding. We perform microseconds resolution single-molecule FRET (smFRET) measurements on four primer templates of 14-15 base pairs and different polarities. Data are analyzed using both second- and fourth- order time correlation functions. At the current stage of this project, our results indicate at least three different conformational stages for gp32 binding. Further analysis is required to compare if and how gp32 dimer bind differently on the different constructs.

Understanding the mechanisms of gp32 filament assembly and sliding on ssDNA templates of known length and polarity

Presenter(s): Megan Barney

Faculty Mentor(s): Andrew Marcus

Poster 78

Session: Sciences

DNA replication is a core biological process that rapidly occurs in both eukaryotic and prokaryotic cells with extreme precision. Gene product 32 (gp32) is a ssDNA binding protein that is important in theT4 bacteriophage DNA replication complex. gp32 is known to bind cooperatively spanning 7 nucleotides of ssDNA. Not only is it known to bind, but it has the ability to unbind from regions of exposed ssDNA during DNA synthesis. This thesis reports microsecond single-molecule FRET (smFRET) measurements on Cy3/Cy5-labeled primer- template (p/t) DNA constructs with and without an addition of 0.5uM gp32. The measurements obtained report the distance between the chromophores that are used to label the ends of 14 and 15 nucleotide segments of ssDNA attached to a p/t DNA construct. These distance measurements can track the conformational changes seen between protein bound vs. unbound states on the microsecond time scale. To analyze the data, a multipoint time correlation function analysis is utilized in order to compare the revealed kinetics of the possible conformational adaptation experienced by the ssDNA of interest. The results of our analysis demonstrate that both length and polarity of the ssDNA influence the way in which gp32 interacts with the ssDNA. Therefore, this SSB is likely to play a critical role at the replication fork during DNA synthesis.

Characterizing the Conformational Fluctuations of DNA Under Physiological and Salt-Stabilized Conditions

Presenter(s): Maya Pande—Biochemistry, Political Science

Co-Presenter(s): Anabel Chang

Faculty Mentor(s): Andrew Marcus

Session: Prerecorded Poster Presentation

The Marcus Group conducts studies on the dynamics of macromolecules in biological environments . In our experiments, we used a variety of techniques to analyze the structure of DNA with the overall goal of better understanding the conformations it can take . Our studies were focused in two areas: (1) understanding the mechanisms of DNA breathing, and (2) conducting experiments on the stabilizing and destabilizing properties of salt solutions on DNA . Techniques included circular and linear dichroism, UV-Vis spectroscopy, and Förster Resonance Energy Transfer (FRET) . Determining the structure of DNA is crucial to understanding biochemical and molecular events essential for gene expression and DNA replication . For these processes to occur, various proteins must access ssDNA coding templates which are otherwise inaccessible due to complementary base pairing in dsDNA . Proteins rely on thermal fluctuations in the DNA double-stranded region at physiological temperatures known as DNA ‘breathing’ . Studies are ongoing, but thus far have led us to a better understanding of the energetic favorability of various conformations of DNA .

Characterizing the Conformational Fluctuations of DNA Under Physiological and Salt-Stabilized Conditions

Presenter(s): Anabel Chang—Biochemistry

Co-Presenter(s): Maya Pande

Faculty Mentor(s): Andrew Marcus

Session: Prerecorded Poster Presentation

The Marcus Group conducts studies on the dynamics of macromolecules in biological environments . In our experiments, we used a variety of techniques to analyze the structure of DNA with the
overall goal of better understanding the conformations it can take . Our studies were focused in two areas: (1) understanding the mechanisms of DNA breathing, and (2) conducting experiments on the stabilizing and destabilizing properties of salt solutions on DNA . Techniques included circular and linear dichroism, UV-Vis spectroscopy, and Förster Resonance Energy Transfer (FRET) . Determining the structure of DNA is crucial to understanding biochemical and molecular events essential for gene expression and DNA replication . For these processes to occur, various proteins must access single- stranded DNA coding templates which are otherwise inaccessible due to complementary base pairing in double-stranded DNA . Proteins rely on thermal fluctuations in the DNA double-stranded region at physiological temperatures known as DNA ‘breathing .’ Studies are ongoing, but thus far have led us to a better understanding of the energetic favorability of various conformations of DNA .