Research Resolves a Mystery in DNA Replication Process

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.

About UMDNJ-ROBERT WOOD JOHNSON MEDICAL SCHOOL

As one of the nation’s leading comprehensive medical schools, UMDNJ-Robert Wood Johnson Medical School is dedicated to the pursuit of excellence in education, research, health care delivery, and the promotion of community health. In cooperation with Robert Wood Johnson University Hospital, the medical school’s principal affiliate, they comprise New Jersey’s premier academic medical center. In addition, Robert Wood Johnson Medical School has 34 other hospital affiliates and ambulatory care sites throughout the region.

As one of the eight schools of the University of Medicine and Dentistry of New Jersey with 2,800 full-time and volunteer faculty, Robert Wood Johnson Medical School encompasses 22 basic science and clinical departments, hosts centers and institutes including The Cancer Institute of New Jersey, the Child Health Institute of New Jersey, the Center for Advanced Biotechnology and Medicine, the Environmental and Occupational Health Sciences Institute, and the Stem Cell Institute of New Jersey. The medical school maintains educational programs at the undergraduate, graduate and postgraduate levels for more than 1,500 students on its campuses in New Brunswick, Piscataway, and Camden, and provides continuing education courses for health care professionals and community education programs. To learn more about UMDNJ-Robert Wood Johnson Medical School, log on to rwjms.umdnj.edu. Find us online at www.Facebook.com/RWJMS and www.twitter.com/UMDNJ_RWJMS.