Chemistry Reference
In-Depth Information
thermodynamically stable, duplex DNA. Recent crystal structures provide valuable
insights, but exactly how this is achieved is not known.
We have developed new single-molecule
fluorescence assays to study various
aspects of a helicases. smFRET approaches have already revealed many surprises in
what was considered the simplest of helicases, E. coli Rep helicase. For example, we
discovered that DNA unwinding catalyzed by Rep stalls if one of the two monomers
within the functional unit dissociates and that unwinding reinitiates only if another
protein is added to reassemble the functional unit [48]. In addition, we discovered that
a Rep monomer undergoes a series of acrobatic movements to reinitiate ssDNA
translocation when it encounters a physical blockade [38].
11.3.1
Helicase: Essential Motor Proteins on the Nucleic Acid Highway
Since its original discovery in 1976 as the DNA unwinding enzyme [61, 62], it has
become clear that helicases participate in virtually all cellular processes that involve
nucleic acid. This includes DNAmetabolism, such as replication, recombination and
repair, as well as RNA processing, such as ribosome assembly, RNA interference,
translation initiation and mRNA splicing [63
-
68]. Helicases are found in all
three kingdoms of life and are extremely numerous: 1
-
2% of eukaryotic genes
are helicases. Helicase studies have important biomedical implications. Several
severe human genetic diseases (Xeroderma pigmentosum, Werners, Blooms,
and Rothmund
-
Thomson syndromes) have been linked to mutations in heli-
cases [69
-
73]. In particular, Werners syndrome results in premature aging as well
as a high propensity to cancer. Since DNA replication and repair are fundamental to
cell growth in all organisms, an understanding of such a basic process as enzyme-
catalyzed DNAunwinding will undoubtedly have an impact on our understanding of
some cancers that result from defects in replication or repair.
The most fundamental property of all helicases is their translocation ability to
move along nucleic acids [74]. This translocation is powered by ATP hydrolysis, hence
helicases are motor proteins. How this motor works remains a mystery. Even the
most elementary issue of the step size of translocation is not yet clear. Also unknown
is why some helicases move in one direction on ssDNA while others move in the
opposite direction. For example, Rep helicase is a 3
0
-5
0
ssDNA translocase and its
DNA unwinding activity requires a 3
0
ssDNA tail on the DNA. Unwinding and
translocation are two de
ning features of helicase, but their relationship is poorly
understood. Helicase must couple its conformational changes resulting from ATP
binding and hydrolysis to its unwinding and translocation action, but exactly how
this is achieved is not known. The long-term goal of our efforts is to measure
helicase function and structural changes simultaneously to probe the core of the
structural
function relationship.
We are using E. coli Rep helicase as a model system for this type of
uorescence
study since (i) its crystal structure is known allowing the rational choice of
labeling sites [75], (ii) its structural homolog PcrA has been crystallized in a number
of different states regarding bound DNA and nucleotides [76], (iii) extensive
-
