de la Rosa, Mario A. Diaz

Koslover, Elena F.

Mulligan, Peter J.

Spakowitz, Andrew J.

Elsevier - Publisher Connector 

Monday, February 22, 2010 221aThe rotor bead technique introduced here allows tracking of the complete
three-dimensional trajectory of a dsDNA translocase in action. It also permits
the application of torque in a laser tweezers apparatus using commercially-
available microspheres.
1147-Plat
Reeling in DNA One Base at A Time: pcrA Translocation Coupled to DNA
Looping Dismantles RecA Filaments
Jeehae Park1, Sua Myong1, Anita Niedziela-Majka2, Jin Yu3,
Timothy M Lohman4, Taekjip Ha1,5.
1Univeristy of Illinois at Urbana Champaign, Urbana, IL, USA, 2Washington
University School of Medicine, St. Louis, IL, USA, 3University of California
at Berkeley, Berkeley, CA, USA, 4Washington University School of
Medicine, St. Louis, MO, USA, 55Howard Hughes Medical Institute, Urbana,
IL, USA.
The mechanism of helicase translocation on DNA remains controversial and
the translocase activity driving their non-canonical functions such as protein
displacement is poorly understood. Here, we used single molecule fluorescence
assays to study a prototypical superfamily 1 helicase, Bacillus stearothermo-
philus PcrA, and discovered a progressive looping of ssDNA that is tightly cou-
pled to PcrA translocation on DNA. Variance analysis of hundreds of looping
events by a single protein demonstrated that PcrA translocates on ssDNA in
uniform steps of 1 nt, reconciling discrepancies in previous structural and bio-
chemical studies. On the forked DNA, rather than acting on the leading strand
to unwind the duplex, PcrA anchored itself to the duplex junction and reeled in
the lagging strand using its 3’-5’ translocation activity. PcrA maintained the
open conformation, not the closed conformation observed in crystallographic
analysis, during looping-coupled translcation. This activity could rapidly
dismantle a preformed RecA filament even at 1nM PcrA, suggesting that the
translocation activity and structure-specific DNA binding are responsible for
removal of potentially deleterious recombination intermediates.
1148-Plat
Protein-Mediated DNA Loops are Resistant to Competitive Binding
Joel D. Revalee, Jens-Christian Meiners.
University of Michigan - Ann Arbor, Ann Arbor, MI, USA.
The lac Repressor protein (LacI) is a canonical genetic regulatory protein. It re-
presses transcription of the lac operon in E. Coli by simultaneously binding to
two distant operator sites on the bacterial DNA and bending the intervening
DNA into a loop. A set of substrate DNA constructs with intrinsic A-tract bends
have been engineered by Mehta and Kahn, which were optimized to form hy-
perstable loops. We present single-molecule measurements of LacI-mediated
loop formation and breakdown rates on these optimized DNA constructs and
demonstrate that repeated formation and breakdown of the loops does not cease
in the presence of 100 nM of free competitor DNA. While this observation
dovetails with bulk competition assays in which the presence of competitor
DNA disrupts the looped complexes only very slowly, our measured loop life-
times of minutes disagree with an inferred lifetime of days from the bulk
assays. We conclude that the LacI-DNA complex can exist in some non-looped
conformation, which can re-loop, but is unexpectedly resistant to competition.
We discuss possible scenarios for such a conformation in light of the data.
1149-Plat
Single Molecule Analysis of Substeps in the Mechanochemical Cycle of
DNA Gyrase
Aakash Basu1, Lena Koslover1, Allyn Schoeffler2, Elsa Tretter2,
James M. Berger2, Andrew J. Spakowitz1, Zev D. Bryant1.
1Stanford University, Stanford, CA, USA, 2University of California
Berkeley, Berkeley, CA, USA.
DNA gyrase is a molecular motor that harnesses the free energy of ATP hydro-
lysis to introduce negative supercoils into DNA. We have characterized the
structural dynamics of processive supercoiling using a real-time singlemolecule
assay in which DNA gyrase activity drives the directional, stepwise rotation of
a submicron rotor bead attached to the side of a stretched DNA molecule. We
are able to directly observe rotational pauses corresponding to rate-limiting
kinetic steps under varying [ATP], and have used simultaneous measurements
of DNA twist and extension in order to characterize transient supercoil trapping
and DNA compaction during the reaction cycle. We have mapped out structural
intermediates of the DNA:gyrase complex on a twist-extension plane, and have
characterized transitions between these states driven by chemical events such
as the cooperative binding of ATP. These measurements motivate several
revisions to previous models based on lower resolution assays [1], and we will
present our results in the context of a new branched kinetic model for the mech-
anochemical cycle. We are now using theoretical calculations together with
measurements of force-dependent changes in extension in order to test specific
geometric models for structural intermediates, and we have begun to analyzestructure-function relationships using single-molecule analysis of gyrase frag-
ments.
[1] Jeff Gore, Zev Bryant, Michael D. Stone, Marcelo Nollmann, Nicholas R.
Cozzarelli, Carlos Bustamante, "Mechanochemical analysis of DNA gyrase
using rotor bead tracking", Nature, 439 (2006)
1150-Plat
Two Structurally Different Families of DNA Base Excision Repair (BER)
Proteins Diffuse Along DNA to Find Intrahelical Lesions
Andrew R. Dunn1, Jeffrey P. Bond1, David M. Warshaw1,
Susan S. Wallace1, Neil M. Kad2.
1The University of Vermont, Burlington, VT, USA, 2The University of Essex,
Colchester, United Kingdom.
Base excision repair (BER) proteins, endonuclease III (Nth) and VIII (Nei)
from E. coli represent two distinct glycosylase families, which recognize and
remove damaged DNA bases. One mechanism by which these glycosylases
scan for DNA lesions is through a simple, one-dimensional diffusive search.
To characterize this search mechanism, we have developed a single molecule
assay in near TIRF to image Qdot-labeled, His-tagged Nth and Nei proteins in-
teracting with YOYO-1 stained l-DNA molecules elongated by hydrodynamic
flow between 5mm silica beads. With an in vitro glycosylase activity assay, we
confirmed that neither YOYO-1 stained DNA nor Qdot labeling significantly
affects glycosylase activity. By imaging individual DNA "tightropes", we ob-
served Qdot-labeled glycosylases interacting with DNA by either binding to or
diffusing on DNA. With increasing ionic strength (50-500mM Kglutamate),
although fewer glycosylases interacted per unit length of DNA, a greater frac-
tion diffused along the DNA. At physiological ionic strength, (150mM KGlu)
both Nth and Nei scan DNA for as much as 10 sec with a diffusion constant of
~1.5 x 105 bp2 sec1, approaching the theoretical limit of rotational diffusion
about the DNA helix. At these rates, the activation barrier for rotational diffu-
sion of 0.7 KbT is slightly below the maximum of ~2 KbT for efficient target
location. We observe no significant difference between Nth and Nei in the rate
or mode of their DNA lesion search mechanism. Interestingly, at elevated ionic
strengths, both families of glycosylases scan above the theoretical limit for free
rotational diffusion (>5 x 105 bp2 sec1). Therefore, the DNA/glycosylase
interface may be optimized for physiological ionic strength, above which the
glycosylase search mechanism shifts from rotational diffusion to a one-
dimensional diffusion without rotation.
1151-Plat
Target-Site Search of DNA-Binding Proteins
Mario A. Diaz de la Rosa, Elena F. Koslover, Peter J. Mulligan,
Andrew J. Spakowitz.
Stanford University, Stanford, CA, USA.
Gene regulatory proteins find their target sites on DNA remarkably fast; the
experimental binding constant for lac repressor is three orders of magnitude
higher than predicted by free diffusion alone. It has been proposed that nonspe-
cific binding aids the search by allowing proteins to slide and hop along DNA.
We develop a reaction-diffusion theory of protein translocation that accounts
for transport both on and off the strand and incorporates the physical conforma-
tion of DNA. For linear DNA modeled as a wormlike chain, the distribution
of hops available to a protein exhibits long, power-law tails. As a result, the
long-time displacement along the strand is superdiffusive. Our analysis predicts
effective superdiffusion coefficients for given nonspecific binding and unbind-
ing rate parameters. Translocation rates experience a maximum with salt con-
centration (i.e., binding rate constant), which has been verified experimentally.
Simulated protein trajectories on
DNA (see figure) agree with our the-
oretical predictions of superdiffusive
transport. Our analytical theory al-
lows us to predict the binding and
unbinding rate parameters that opti-
mize the protein translocation rate
and the efficiency of the search. Fi-
nally, we use our theory to predict
rates of target site localization under
various experimental conditions.1152-Plat
Illuminating the DNA Binding Behavior of Mitochondrial Transcription
Factor A
Ge´raldine Farge1, Onno D. Broekmans1, Niels Laurens1, Linda Dekker1,
Maria Falkenberg2, Erwin J.G. Peterman1, Gijs J.L. Wuite1.
1VU University Amsterdam, Amsterdam, Netherlands, 2University of
Gothenburg, Gothenburg, Sweden.


Target-Site Search of DNA-Binding Proteins 

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Target-Site Search of DNA-Binding Proteins

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