Effects of macromolecular crowding on DNA structure and protein-mediated looping in DNA. hKIF4a compaction of DNA Público

Abstract

The cellular environment is highly crowded, but many laboratory measurements are

conducted in dilute buffer. In order to begin to bridge the gap between in vitro

and in vivo measurements, macromolecular crowders are often included in in vitro

experiments. An extensive survey of the literature indicated that experiments with

biologically relevant crowders, experiments with mixtures of variously sized crowders,

and experiments elucidating information on the interplay between macromolecular

crowding and liquid-liquid phase separation within the cell all require further work.

Tethered particle motion experiments were designed to directly investigate the effects

of macromolecular crowding on protein-mediated looping in DNA. Macromolecular

crowding caused DNA to preferentially adopt the parallel loop conformation over the

anti-parallel loop conformation. Another set of tethered particle motion experiments

were performed to characterize the compaction of DNA by the protein hKIF4a, a

chromokinesin. hKIF4a required ATP to compact DNA, although a truncated form of

hKIF4a lacking the ATPase domain, compacted DNA better than wild-type hKIF4a

in the presence of AMP-PNP, an non-hydrolyzable ATP analogue.

Table of Contents

1 Introduction 1

2 Effects of macromolecular crowding on DNA structure 4

2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

2.2 Compaction and extension of DNA . . . . . . . . . . . . . . . . . . . 5

2.2.1 Polyethylene glycol . . . . . . . . . . . . . . . . . . . . . . . . 6

2.2.2 Dextran . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

2.2.3 Bovine serum albumin . . . . . . . . . . . . . . . . . . . . . . 14

2.2.4 Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

2.3 Kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

2.3.1 Crowders vs. viscogens . . . . . . . . . . . . . . . . . . . . . . 15

2.3.2 Phase separation . . . . . . . . . . . . . . . . . . . . . . . . . 18

2.4 Protein-DNA interactions . . . . . . . . . . . . . . . . . . . . . . . . 22

2.4.1 Nucleoid-associated proteins . . . . . . . . . . . . . . . . . . . 23

2.5 Theoretical works on effects of macromolecular crowders on polymers 27

3 Effects of macromolecular crowding on LacI-mediated looping in

DNA 30

3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

3.1.1 DNA structure . . . . . . . . . . . . . . . . . . . . . . . . . . 31

3.1.2 lac repressor (LacI) and LacI mediated looping . . . . . . . . 31

3.2 Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . 32

3.2.1 DNA preparation . . . . . . . . . . . . . . . . . . . . . . . . . 32

3.2.2 Chamber preparation . . . . . . . . . . . . . . . . . . . . . . . 32

3.2.3 Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

3.2.4 Tethered particle motion (TPM) microscopy . . . . . . . . . . 34

3.2.5 TPM microscope . . . . . . . . . . . . . . . . . . . . . . . . . 35

3.2.6 TPM data collection and analysis . . . . . . . . . . . . . . . . 37

3.3 Discussion and conclusion . . . . . . . . . . . . . . . . . . . . . . . . 39

4 hKIF4a compaction of DNA 40

4.1 Introduction to hKIF4a . . . . . . . . . . . . . . . . . . . . . . . . . . 40

4.2 hKIF4a utilizes ATP to compact DNA . . . . . . . . . . . . . . . . . 41

4.3 hKIF4a variation without ATPase compacts DNA . . . . . . . . . . . 44

4.4 BSA may aide hKIF4a in compaction of DNA . . . . . . . . . . . . . 45

4.5 Discussion and conclusions . . . . . . . . . . . . . . . . . . . . . . . . 47

5 Conclusion 49

Bibliography 52

About this Dissertation

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School	Laney Graduate School
Department	Physics
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Physics, Molecular
Palabra Clave	Tethered Particle Motion Macromolecular Crowding DNA Single-molecule
Committee Chair / Thesis Advisor	Laura Finzi, Emory University David Dunlap, Emory University
Committee Members	Justin Burton, Emory University Kurt Warncke, Emory University Keith Weninger, North Carolina State University

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