Research From UMDNJ-Robert Wood Johnson Medical School Resolves A Mystery In DNA Replication Process

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PISCATAWAY – 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. This is accomplished as the double helical (coil-shaped) DNA divides into two strands that are then duplicated. New research from UMDNJ-Robert Wood Johnson Medical School and Cornell University identifies how the ring-shaped helicase enzymes that separate the strands of double helical DNA track forward along the DNA without slipping backward.

Ring-shaped helicases are key players in replicating not only the human genome but those of pathogenic viruses (viruses with the ability to cause disease) such as the human papilloma virus (HPV) that causes cervical cancer. It is hoped that understanding how this class of helicases works will pave the way to new therapeutic treatments for human diseases.

The study, “ATP-induced helicase slippage reveals highly coordinated subunits,” was chosen for advanced online publication in Nature this week, and can be found online at: http://www.nature.com/nature/journal/vaop/ncurrent/full/nature10409.html.

To initiate unwinding of DNA, the helicase enzymes rely on the presence of nucleotides (molecules that are basic building blocks of DNA and RNA), generally a nucleotide called adenosine triphosphate or ATP. However, when explicitly examining DNA unwinding with ATP, the research team discovered that the phage T7 helicase unwinds DNA with ATP at a fast rate but it slips repeatedly.

“To our knowledge this is the first direct observation of helicase nucleotide-specific slippage, and our detailed study of this phenomenon reveals a potential mechanism for ensuring successful unwinding and duplication of DNA,” said Smita Patel, PhD, professor of biochemistry at UMDNJ-Robert Wood Johnson Medical School, along with her collaborator Michelle Wang from Cornell University and the Howard Hughes Medical Institute.

However, the researchers found that helicase slippage was stopped when another nucleotide, deoxythymidine triphosphate, or dTTP, was added to ATP, and that mixtures of ATP and dTTP controlled the degree of slippage.

“Through further examination of the DNA unwinding reaction with mixtures of ATP and dTTP, we discovered the mechanism by which the helicase subunits coordinate their activities to ensure efficient strand separation without falling off the DNA,” said Dr. Patel.

The study explains that for a helicase to slip, all six of its subunits must simultaneously lose their grip on the DNA. The presence of dTTP increased the helicases’ ability to bind successfully to DNA, thereby reducing slippage. The team explains that each of the subunits takes a turn in assuming the leading position to pull on the DNA and to move the helicase ring forward. This work reveals that while the leading subunit is pulling on the DNA, the remaining subunits are holding on to the DNA and helping the leading subunit to move forward without falling off the DNA. Holding on to the DNA tightly requires some amount of dTTP, and explains how dTTP prevents helicase slippage. This type of cooperation between the helicase ring subunits makes the helicase effective at unwinding DNA. If the process of DNA unwinding was interrupted by slippage of the helicase, and was left uncorrected, it would stall the replication process causing harm to the normal cell growth.

The research was supported by grants from the National Institutes of Health and the National Science Foundation and Cornell’s Molecular Biophysics Training Grant.


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