Van Oijen visualizes DNA replication of single molecules
By Jeffrey Stumpf
University of Groningen professor Antoine van Oijen, Ph.D., showcased a variety of new imaging techniques that challenges basic beliefs about DNA replication and its response to nucleotide damaging agents. As part of the NIEHS Laboratory of Molecular Genetics (LMG) Fellows’ Invited Series, van Oijen dazzled researchers Sept. 24 with images of DNA replication proteins altering single DNA molecules in real time.
Van Oijen used a variety of imaging techniques of fluorescently labeled proteins to show how simple in vitro replication systems coordinate various DNA modifying activities, such as the unwinding of DNA and priming DNA for replication. Single molecule imaging of DNA replication in E. coli and Xenopus cell extracts demonstrated important fundamental results about origins of replication and bypass of damage.
Trained as a physicist, van Oijen’s research is a successful marriage of physics, microbiology, biochemistry, and virology. Jessica Williams, Ph.D., research fellow in the NIEHS DNA Replication Fidelity Group hosted the speaker’s visit and noted the importance of the contributions from a variety of different fields. “By bringing these disciplines together, it has really provided novel answers to long-standing biological questions,” Williams commented.
Visualizing DNA replication loops
Van Oijen described a technique that used modified viral DNA, with one end attached to a glass coverslip and the other end attached to a small bead, that can be visualized. When a force is applied to the DNA, the rate at which the DNA stretches to different lengths is different, depending on whether it is double-stranded (dsDNA) or single-stranded (ssDNA). Thus, van Oijen can determine the precise rates that polymerases create, and helicases unwind, double-stranded DNA.
For many NIEHS scientists in the audience, the possibilities, using van Oijen’s technology, are endless. “This new imaging technique is really a breakthrough in the field,” Williams stated. “It is one of the first demonstrations of single-molecule fluorescence techniques to study protein complexes at their physiologically relevant concentrations.”
The single-molecule studies became more complex, when more replication proteins were added to the assay. Van Oijen recounted a particular moment of disappointment when adding the single-stranded binding protein gp 2.5 to the complex caused the length of the ssDNA-bound complex to be the same length as dsDNA.
“This was two or three years after we established the assay that we realized that the assay doesn’t work,” lamented van Oijen.
The potential letdown of wasting years of research was short-lived, as van Oijen realized that his assay was actually the first to allow detection of replication loops that grow during lagging strand synthesis. Van Oijen showed that the replication of the previous fragment, and the priming of the next Okazaki fragment, signal the release of the loop.
Seeing polymerase degradation is believing
Van Oijen addressed the long-standing question of how specialized DNA polymerases, that function in DNA repair, gain access to the replication fork. By using fluorescently labeled proteins in E. coli, van Oijen imaged a striking response to UV damage. Pol III polymerase that is responsible for normal DNA replication disappeared, and the Pol V polymerase that bypasses UV-induced damage saturated the cell.
These results suggest that Pol V is eliminating the competition at the replication fork. In fact, van Oijen demonstrated the degradation of Pol III by a Pol V subunit, accounting for the disappearance of the Pol III signal. While previous genetic evidence suggested interplay between Pol III and Pol V at a UV-induced lesion (see related story), van Oijen’s microscopy data, and the degradation of Pol III, changes the current thinking about the interplay among DNA polymerases.
Mutations in the Pol V human homolog cause a variant of xeroderma pigmentosa, a rare genetic disease that causes extreme sensitivity to sunlight and predisposition to skin cancer. Microscopy approaches may also illuminate interactions among human polymerases, to help explain the mechanisms that occur to prevent disease.
(Jeffrey Stumpf, Ph.D., is a research fellow in the NIEHS Laboratory of Molecular Genetics Mitochondrial DNA Replication Group.)