DNA replication is a basic function of living organisms, allowing cells to divide and multiply, all while maintaining the genetic code and proper function of the original cell. The process, or mechanism, by which this is accomplished presents many challenges as the double helical (coil-shaped) DNA divides into two strands that are duplicated by different methods, yet both strands complete the replication at the same time.
New research by a team from UMDNJ-Robert Wood Johnson Medical School in conjunction with the University of Illinois and published in the Dec. 17 issue of Nature, has addressed this fundamental problem. The study identifies three essential ways the synthesis of the two strands is coordinated by enzymes, settling scientific deliberations on how the two DNA strands are copied in the same time span.
"DNA replication is a fundamental reaction required for the maintenance, survival, and propagation of living cells. It is also a very complex reaction that has been studied for decades without a clear understanding of how the two interwound strands are copied at the same time," says Smita Patel, PhD, professor of biochemistry at Robert Wood Johnson Medical School and lead author of the paper. "Our study explains how the replication is coordinated -- an important piece of the puzzle, because errors in DNA replication can cause disabilities and disease, such as cancer."
The helicase enzyme initiates DNA replication, by unwinding, or separating, the strands which are then reproduced by polymerase enzymes which are responsible for making an exact copy of the DNA. One strand, called the leading strand, is reproduced continuously, whereas the other, lagging strand is reproduced in fragments that are later joined together. How the two strands are replicated at the same time was not previously understood because the polymerase enzyme that replicates the lagging strand must recycle after the completion of each fragment.
According to Dr. Patel, the researchers used these state-of-the-art methods to measure the progression of DNA synthesis in the millisecond time scale. "We employed rapid kinetic methods to investigate this problem and coupled it with single molecule fluorescence measurements to show that the replication enzymes do not pause, as previously thought, but our studies suggest that the short fragments are synthesized at a slightly faster rate so lagging strand synthesis can keep up with the synthesis of the leading strand that is made continuously," said Dr. Patel.
These methods captured the replication enzymes in the act of making the DNA and identified the three ways the strands complete replication simultaneously. First, as Dr. Patel noted, the lagging strand polymerase keeps up with the leading strand polymerase by moving a little faster, which gives the lagging polymerase the extra time it needs to recycle and start the synthesis of a new DNA fragment. This finding supports an early model proposed by Bruce Alberts, a professor emeritus in the department of biochemistry and biophysics at the University of California, San Francisco, former president of the National Academy of Sciences and editor-in-chief of Science magazine.
The study also shows that the reproduction time is further reduced by making the RNA primer ahead of time as the lagging-strand synthesis progresses through the cycle. The RNA primer is a sequence of nucleotides (molecules that, when joined together, make up the structural units of RNA and DNA) copied from DNA. According to Dr. Patel, the polymerase needs RNA primer to initiate replication of a new fragment and that making it "on the fly" saves time in the replication process. Lastly, the research shows that the RNA primer is kept in physical proximity to the lagging strand polymerase by means of a priming loop so that the polymerase enzyme can access it and begin replication of a new fragment quickly.
Thus, the faster movement of the lagging strand polymerase enzyme, the ability to make the RNA primer ahead of time and the ability for the polymerase enzyme to access the RNA primer quickly due to its close location allow the two strands of the DNA to be copied in the same time span.
The study was a collaboration of investigative teams led by Smita Patel, PhD, professor of biochemistry at Robert Wood Johnson Medical School and Taekjip Ha, PhD, HHMI investigator and professor of physics and a co-director of Center for the Physics of Living Cells at the University of Illinois at Urbana-Champaign. The study, officially titled "Coordinating DNA replication via priming loop and differential synthesis rate" was chosen for advanced online publication in November and appears in the December 17 print issue of Nature, pages 940-944. The first author of the paper is Manjula Pandey, PhD, a research teaching specialist and additional authors include graduate student Ilker Donmez and research teaching specialist Gayatri Patel of the department of biochemistry at Robert Wood Johnson Medical School and Salman Syed, research scientist in the department of physics at the University of Illinois at Urbana-Champaign.
The research was supported by grants from the National Institutes of Health and the National Science Foundation.
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