BIn biology classes, students are taught that once a cell’s replication machinery is aligned, DNA polymerase sets off along the double helix, replicating the strand continuously, like a car on a highway. When an error occurs, the same enzyme stops, reverses itself, corrects the mistake, and continues its journey to the end of the line.
but, Published research In the last 10 to 15 years, increasingly challenged This model suggests that multiple polymerases are involved in DNA replication and proofreading.1, 2 Now, in a publication nature communications, The Vrije Universiteit Amsterdam team provided the following additional evidence: DNA polymerase DNA does not replicate as continuously as once believed.3
“In contrast to these proteins being very stably bound on the DNA, they move back and forth all the time,” he said. antoine van oyena molecular biophysicist at the University of Sydney, was not involved in the study. “It’s like changing a tire while driving.” Understanding the activity of DNA polymerases will help scientists better understand DNA replication and repair and how these processes can go wrong. This is useful for investigating whether it can lead to diseases such as cancer.
“This discovery [happened] It’s just a coincidence,” he said. Xu Longfubiophysicist and postdoctoral researcher Gis WiteLaboratory at Vrije Universiteit Amsterdam. The research team initially set out to study another replication machinery protein, single-stranded DNA-binding protein, and its interaction with DNA polymerase. But they first needed to establish how the two proteins interact independently with DNA. While investigating the activity of DNA polymerase, the team observed something unexpected. This means that proteins can rapidly jump onto and detach from nucleic acids.
To find out where DNA polymerases are located and what they do, Xu and colleagues combined two methods: confocal microscopy and optical tweezers. The researchers stretched an 8,000 kilobase DNA strand between two laser-fixed beads. The tethered DNA strands consisted of double-stranded (dsDNA) segments that became single-stranded (ssDNA). The researchers experimentally stimulated these enzyme functions by using a laser to apply varying amounts of force to mimic the tension that DNA normally experiences during replication and proofreading. Next, by adding a fluorescent tag to the DNA polymerase, they tracked the enzyme’s progress and binding dynamics to DNA.
“You can apply tension to DNA and you can visualize the movement of polymerase on DNA, but these two datasets are independent. We wanted to correlate them.” Mr. Schuh said. However, he explained that synchronizing these two datasets and mapping the protein’s path along the chain poses challenges. But by tracking the binding of fluorescent proteins at dsDNA-ssDNA junctions, the researchers were able to overlay these two pieces of information to reveal the behavior of DNA polymerases.
The researchers observed that, on average, a single DNA polymerase molecule remained bound to the nucleic acid at the junction for just over a second. This is far from the continuous combination described in most textbooks. In further contrast to established theory, during this period, single enzymes performed only either elongation or proofreading, and in some cases stopped processing DNA as well. Rather than backing up to correct the error, the enzyme separated from the nucleic acid and attached another.
“The idea of installing the motor backwards seems very appealing to us, but it’s much more efficient to throw away the motor,” Wuite explained. Unlike cars, cells have multiple DNA polymerase motors, allowing enzymes that already have the necessary configuration to bind to DNA and correct errors to take over. This exchange requires less energy than changing the structure of the same protein to perform a different function.
But because the DNA polymerase activity appeared to be seamless and uniform, the researchers thought there was a process in which one enzyme picks up another where it left off, acting like a memory. . They analyzed a single elongation event and observed that the polymerase unbound and rebound multiple times, but each time it resumed the same function.
To study this further, the team evaluated the active state (enzymatic or paused) of the fluorescent polymerase before, during, and after binding to DNA through several experiments. They found that the most common pattern was that the activity was the same at all three observation points, regardless of whether the enzymatic period was during exonuclease repair or DNA elongation.
“This experiment really puts the nail in the coffin of this model where everything is stable in the DNA,” van Oyen said. He added that structural studies are important to add additional context to these mechanisms.
“The real dream, of course, is to simultaneously observe the various components at work at the junction where single- and double-stranded DNA meet,” Witte said. Xu and his colleagues began working on these experiments.
Researchers are applying this dual approach to other problems, such as studying chromosome segregation. “Most of what you read about the makeup of chromosomes in biology books is fantasy,” Witte says. “With our tool, you can actually take some steps forward, and in fact, understanding how the tool is actually configured can help you understand what will work or fail. You can understand some of the basics.”
Conflict of Interest Disclosure: Gijs Wuite is a co-founder and shareholder of the biological research equipment company LUMICKS and holds a conflict of interest patent. method and technology Explained in this story.
