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Helicases are a special category of molecular motors that modify DNA (deoxyribonucleic
acid, the fundamental building block of genes and chromosomes). They
do so by moving along strands of DNA, much the same way cars move on
roads, using an energy-packed molecule, adenosine triphosphate (ATP)
as a fuel source. Their primary function is to unzip double-stranded
DNA, allowing replication and repair of the strands.
DNA is a fragile molecule that undergoes dramatic changes when exposed
to radiation, ultraviolet light, toxic chemicals or byproducts of
normal cellular processes. DNA damage, if not repaired in time, may
lead to mutations, cancer or cell death. Many helicases in the Rad3
family are key players in the cell�s elaborate machinery to prevent
and repair such damage. Mutations in the human members of this
helicase family impede DNA repair and may contribute to breast cancer,
Fanconi Anemia and Xeroderma pigmentosum.
The researchers studied the archaeal version of RAD3.
Archaea are microbes whose DNA repair systems are closely related to
those of human cells.
�(The archaeal Rad3) is a very good representative of a unique family
of structurally related DNA repair helicases, all of which have the
same motor core and share an unprecedented (for helicases) structural
feature � an accessory domain stabilized by an iron-sulfur cluster,�
Spies said.
Working with archaea has the advantage of allowing the researchers to
increase the amount available protein and also permits easy genetic
manipulation.
Like other helicases, Rad3 is composed of a chain of amino acids. It
also contains an ancient prosthetic group called an iron-sulfur
cluster, an assembly of four iron and four sulfur atoms incorporated
into the protein structure through interaction with four cysteine
residues of the amino acid chain.
"DNA helicases, which belong to the Rad3 family, have an auxiliary
domain inserted within a conserved motor core. The structure of this
domain is stabilized by an iron-sulfur cluster, whose integrity seems
to be essential for proper function of these enzymes in DNA repair,"
Spies said. By mutating the cysteine ligands to the cluster, the
researchers probed its role in the molecular mechanism of Rad3 enzymes.
Some of these mutations uncoupled DNA translocation and ATP hydrolysis,
meaning that the engine of the protein could still use the ATP fuel
but was no longer capable of moving along the DNA.
This analysis also revealed that the integrity of the cluster and the
iron-containing domain is crucial for recognition of specific DNA
structures believed to be physiological targets for this helicase. �On
making these mutations, the helicase no longer behaves like it�s
supposed to,� said graduate student Robert Pugh, lead author on the
study. �The cluster is still there but the environment around it is
somehow changing.�
This research was performed in collaboration with Isaac Caan�s group
from Animal Sciences whose lab is engaged in the study of nucleotide
metabolism in archaea.
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