Genetic Repair Mechanism Clears The Way For Sealing DNA Breaks

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Genetic Repair Mechanism Clears The Way For Sealing DNA Breaks

http://www.medicalnewstoday.com/medicalnews.php?newsid=54677

Scientists investigating an important DNA-repair enzyme now have a

better picture of the final steps of a process that glues together,

or ligates, the ends of DNA strands to restore the double helix.

The enzyme, DNA ligase, repairs the millions of DNA breaks generated

during the normal course of a cell's life, for example, linking

together the abundant DNA fragments formed during replication of the

genetic material in dividing cells.

" Our study shows that DNA ligase switches from an open, extended

shape to a closed, circular shape as it joins DNA strands together, "

says the study's senior author Tom Ellenberger, D.V.M, Ph.D., the

H. Wittcoff Professor and head of the Department of

Biochemistry and Molecular Biophysics at Washington University

School of Medicine in St. Louis. " The ligase resembles a wristwatch

that latches around the DNA ends that are being joined. "

DNA is surprisingly reactive and under continuous assault from

environmental toxins and reactive cellular metabolites. A means of

repairing DNA damage is vital to maintaining the integrity of the

genetic blueprint.

When these repair processes go awry, cells can malfunction, die or

become cancerous, so researchers would like to know how " DNA

mechanics " do their jobs. DNA ligases are attractive targets for the

chemotherapy of cancer and other diseases.

DNA ligase works in concert with another ring-shaped protein known

as a sliding clamp. Sliding clamps, such as the human PCNA protein,

are master regulators of DNA repair, providing docking sites that

recruit repair enzymes to the site of damage.

" When ligase stacks against PCNA and encircles the DNA, we think

this interaction ejects other repair proteins from PCNA, " says

Ellenberger. " In this role, ligase may serve as the final arbiter of

DNA repair, certifying that the DNA is in pristine condition and

ready for the final step of DNA end joining. "

In this study of DNA ligase, published in the Oct. 20 issue of

Molecular Cell, Ellenberger's research group teamed with scientists

from The Scripps Research Institute (TSRI), the University of

land School of Medicine and Lawrence Berkeley National

Laboratory (LBNL).

To visualize the complicated and dynamic structures of DNA ligase

and PCNA, both separately and in a complex, Ellenberger and his

group worked closely with LBNL scientists to take advantage of the

intense X-rays and advanced technologies of the SIBYLS synchrotron

beamline at the Berkeley lab Advanced Light Source.

The researchers used a combination of X-ray crystallography and

small angle X-ray scattering (SAXS). They conducted their studies

with a model organism called Sulfolobus solfataricus that has many

of the same biochemical characteristics of multicelled organisms,

including humans.

" We expected that DNA ligase would latch shut when bound to the ring-

shaped PCNA protein, " says Ellenberger. " However, the SAXS

experiment clearly shows that ligase remains in an open conformation

enabling other repair proteins to bind PCNA until the DNA is engaged

and ligase snaps shut. "

Co-author Tainer, Ph.D., professor at LBNL and TSRI, says the

results reveal for the first time how these proteins can dynamically

assemble and change their shape to join DNA ends during replication

and repair.

The closed conformation of DNA ligase bound to DNA was imaged in a

separate study previously reported by Ellenberger's group.

Ellenberger says that the challenge for the future is to study the

molecular choreography of ligase, PCNA and DNA in the same

experiment, which will require new methods of analyzing the SAXS

data.

" The SAXS methods offer a powerful means of visualizing large

proteins and protein complexes that are difficult or impossible to

crystallize, " says Ellenberger. " Imaging of complex processes will

require a variety of tools that address different levels of

biological organization from the molecular level to whole animals. "

Research on biological imaging is one aspect of the University's

BioMed21 initiative, which calls for converting knowledge of genetic

mechanisms into practical applications.