rainBiology classes teach students that when the cell’s replication machinery works together, DNA polymerase travels down the double helix like a car on the highway, replicating successive strands. When an error occurs, the same enzyme stops, reverses, corrects the error, and then returns to constant movement to the end of the line.
but, published research Increasingly over the past 10-15 years challenge The model suggests that multiple polymerases are involved in DNA replication and proofreading.1,2 Now, in a publication nature Communications, A team from Vrije University Amsterdam provided further evidence: DNA polymerase We do not replicate our DNA continuously, as was once believed.3
“Unlike proteins, which are very stably bound to DNA, they are always going back and forth,” he said. Antoine van OizenThe molecular biophysicist at the University of Sydney was not involved in the study. “It’s almost like changing a tire while driving.” Unraveling the activity of DNA polymerase could help scientists better understand DNA replication and repair and explore how these processes fail and lead to diseases such as cancer.
“This discovery [happened] “By chance,” he said. Xu LongfuI am a biophysicist and postdoctoral researcher. Gis WitteLaboratory at Vrije University Amsterdam. The team initially began studying its interactions with another replication machinery protein, single-stranded DNA-binding protein and DNA polymerase. But they first needed to determine how the two proteins interact independently with DNA. While investigating DNA polymerase activity, the research team discovered something unexpected. In other words, proteins are moving around quickly in nucleic acids.
To determine where DNA polymerase is and what it is doing, Xu and his colleagues combined two methods: confocal microscopy and optical tweezers. The research team stretched an 8,000 kilobase strand of DNA between two beads held in place by a laser. The joined DNA strands consisted of double-stranded (dsDNA) segments that became single-stranded (ssDNA). Using a laser, they applied varying amounts of force to mimic the tension DNA typically experiences during replication or proofreading to experimentally promote these enzymatic functions. They then added a fluorescent tag to the DNA polymerase to track the enzyme’s progress and binding dynamics to DNA.
“We can apply tension to the DNA and visualize the polymerase movement of the DNA, but these two data sets are independent. We wanted to relate them,” Xu said. But he explained that synchronizing these two data sets and mapping the protein’s path along the strand is a challenge. But by tracking fluorescent protein binding at the dsDNA-ssDNA junction, the team was able to overlap these two pieces of information to reveal the behavior of the DNA polymerase.
The researchers observed that, on average, a single DNA polymerase molecule remains bound to the nucleic acid at the junction for more than one second. This is a far cry from the serial joins described in most textbooks. In further contrast to this doctrine, during this period single enzymes performed only extensions or proofreading, sometimes pausing even on the DNA. Instead of backing up to correct the error, the enzyme separates from the nucleic acid and allows another enzyme to bind to it.
“The idea of putting the motor in reverse sounds very attractive to us, but throwing the motor out is much more efficient,” Wuite explained. Unlike cars, cells have multiple DNA polymerase motors, so enzymes that are already in the configuration needed to bind DNA and correct errors can take over. This exchange uses less energy than changing the conformation of the same protein to perform a different function.
However, because the activity of DNA polymerase appeared to be smooth and uniform, the team suspected that a process existed that helped one enzyme pick up where another enzyme left off, acting like a memory. They analyzed one extension event and observed the polymerase unbinding and rebounding several times, but resuming the same function each time.
To study this further, the team assessed the activity state (enzymatic or paused) of the fluorescent polymerase before, during, and after binding to DNA over the course of several experiments. They found that the most common pattern was that activity was the same at all three observation points, regardless of whether the enzyme period was during exonuclease repair or DNA extension.
“This experiment is really the nail in the coffin of this model where everything is stable in the DNA,” van Oijen said. He added that structural studies will be important to add additional context to these mechanisms.
“The real dream, of course, is to work at the intersection where single-stranded and double-stranded DNA meet, seeing all the different components simultaneously,” Wuite said. Xu and his colleagues set out to conduct these experiments.
Researchers are also applying this dual approach to other questions, such as studying chromosome segregation. “What you read in biology books about chromosome organization is largely fantastic,” Wuite said. “We can use the tools to actually take some steps and understand some of the basics of what’s right and what’s wrong by understanding how it’s actually structured.”
Conflict of Interest Disclosure: Gijs Wuite is a co-founder and shareholder of LUMICKS, a biological research equipment company, and holds patents related to: methods and technology It is explained in this story.
