DNA enzyme target exhibits great dexterity
during replication that may serve as a precursor to understanding
genetic disease caused by abnormal enzyme activity.
The discovery could also form a basis for a treatment or gene therapy, which could preserve the integrity of DNA by stripping the DNA strand of potentially toxic proteins that accumulate during replication.
Scientists from the Howard Hughes Medical Institute researchers show that the helicase enzyme, which normally crawls along the length of a DNA strand during its function in replication and other processes, exhibits flexibility when it encounters an obstacle.
The studies show that the enzyme snaps back to its original position on the DNA strand so that it can begin the process again.
Helicases are a critical part of the DNA replication process because they unwind double-stranded DNA to create single strands suitable for copying by the replication machinery.
This and other helicase activity in the cell depends on the ability of the helicase's protein "engine" to crawl along the DNA strand.
The discovery could help those with Bloom syndrome, another helicase-related disease.
It is caused by a defect in a different helicase. People with this defect show an increased propensity for many types of cancer.
The helicase involved in this disorder appears to play a role in the general maintenance of genomic integrity.
If scientists can understand its regular function and how it is altered when mutated, it may be possible to understand a more general mechanism underlying cancer.
In their test tube experiments, Taekjip Ha, an investigator at the University of Illinois Urbana-Champaign, concentrated on the engine component of a helicase called Rep from the bacterium E. coli.
They followed the motion of the Rep engine along the DNA strand by tagging it with a green fluorescent dye.
They also attached a molecule of a red fluorescent dye to the destination end of the DNA strand.
This technique, called fluorescence resonance energy transfer (FRET), enables researchers to determine how the tagged molecules move relative to one another by observing how one dye molecule transfers energy to the other.
As Rep approached a blockaded end of the DNA strand, the researchers expected to see a gradual reduction in the red signal and an increase in the green signal.
"We saw the gradual FRET increase as we expected, and we knew that the protein was moving in a particular direction that would bring the two dyes close to each other," said Ha.
The protein would did not stop at the blockade and fall off the DNA, observing its movement back where it began on the DNA. Then it repeated that gradual movement toward the barrier again and repeated the snap-back movement.
Once it reached the blockade, it stretched out to grab the DNA strand near its starting point, formed a loop in the DNA and then released its stopping point to bring it back to its beginning.
The results suggested that the repetitive shuttling could prevent the accumulation of unwanted proteins on the single-stranded DNA that could prove toxic to the cell.
The researchers likened the process to brushing teeth, with the Rep protein acting like the teeth that cleans the DNA 'floss.'
Ha and his colleagues would next like to explore whether the shuttling phenomenon they observed in their test tube studies actually functions in other helicases and in the living cell.
"There are rare disorders in humans that could possibly involve malfunction of the helicase engine. People with one such disorder, called Werner syndrome, grow normally until they reach adolescence, when they begin to age very rapidly," said Ha.
The rapid aging suggests that helicase may play a role maintaining genomic integrity.
The study is published in the October 27, 2005, issue of the journal Nature.
