The St. Jude researchers developed a 3-D image of a key part of UvsW, a type of enzyme called a helicase. Helicase enzymes open up the double-stranded DNA molecules like a zipper, so each strand can be replicated to produce two new pieces of DNA. T4, a virus that infects bacteria, uses UvsW during a process called recombinant-dependent replication (RDR). RDR is a type of natural genetic engineering by which viruses, plants and animals introduce new genes into the DNA of one chromosome during replication and repair of broken DNA by using a section of another chromosome as a blueprint. The scientists studied the T4 helicase because it is a simple but effective model for understanding how similar helicases perform the same job in human cells.
The finding is important because UvsW is vital to the processes of DNA replication and repair, keeping the genetic material "stable" so mutations do not occur, according to Stephen White, Ph.D., chair of the St. Jude Department of Structural Biology and a member of the Department of Molecular Sciences at the University of Tennessee, Memphis. White is senior author of the Structure report.
UvsW also triggers RDR as part of a rescue mission to repair a snag in DNA replication called a stalled replication fork.
Normally, each single strand of DNA serves as a template, or blueprint, for remaking the other strand. In this way the enzymes involved in DNA replication rebuild each strand to make two chromosomes out of one. When a section of the double-stranded DNA molecule is separated into two single strands, the resulting Y-shaped structure is called the replication fork. A stalled replication fork occurs when the two strands of a chromosome's double-stranded DNA fail to separate. UvsW restarts RDR by unsnarling the stalled fork and restarting replication.
During RDR, the ends of the free strands at the fork drift into the DNA of another chromosome, like the free arm of one person pushing into the folded arms of another person. This causes a single strand from the first chromosome to be used as a blueprint by the second chromosome. By using the invading strand as a template to make new DNA, the second chromosome acquires new genes from the first chromosome, while the first chromosome acquires new genes from the second one. UvsW also orchestrates the use of one strand of DNA as a blueprint to patch up a broken section of another piece of DNA. This also contributes to introducing new genes into chromosomes.
"This process is like the continual mixing up of pieces of blueprints for two different houses," White said. "You still have two sets of blueprints, but each set has plans for one or more rooms that were originally in the other blueprint. The result is that the blueprints keep changing, adding variety to the houses that are made from them."
This is what happens to chromosomes during genetic recombination. The resulting shuffling of genes is the source of the diversity of life among viruses, as well as among plants and animals.
"Now that we know the exact structure of the part of UvsW that interacts with DNA, we can take a closer look at how this important enzyme works," White said. "Stable replication keeps the DNA stable, even while it is undergoing recombination. UvsW plays a major role in keeping DNA stable."
There is evidence to suggest that two human helicases, Bloom and Werner, may have similar roles to those of UvsW in rescuing stalled replication forks. Mutations in these helicases cause Bloom's syndrome and Werner syndrome, rare disorders linked to a predisposition to develop cancer.
White used a modified form of X-ray crystallography to create images of UvsW. In this technique, a crystallized sample of a protein is bombarded with a beam of X-rays. The pattern formed by the diffraction of the beams off the crystal is used to create a computer-generated, 3-D image of the protein. The modified version of this technique, multiwave anomalous diffraction (MAD), also bombards a crystallized protein with X-rays. However, in MAD, the investigator is able to alter the diffraction pattern by first having certain atoms that surround the protein absorb some of the incoming X-rays.
The paper's other authors are E. Allen Sickmier (St. Jude and University of Tennessee) and Kenneth N. Kreuzer (Duke University). This work was supported in part by a Cancer Center (CORE) grant and by ALSAC.
St. Jude Children's Research Hospital
St. Jude Children's Research Hospital is internationally recognized for its pioneering work in finding cures and saving children with cancer and other catastrophic diseases. Founded by late entertainer Danny Thomas and based in Memphis, Tennessee, St. Jude freely shares its discoveries with scientific and medical communities around the world. No family ever pays for treatments not covered by insurance, and families without insurance are never asked to pay. St. Jude is financially supported by ALSAC, its fundraising organization. For more information, please visit www.stjude.org.
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Structure