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Whole genome sequencing illuminates lagging strand DNA replication

By Jeffrey Stumpf
December 2010

Thomas Kunkel, Ph.D.
Kunkel worked with current and former members of his group, as well his LMG colleagues Principal Investigator Mike Resnick, Ph.D., and Senior Staff Scientist Dmitry Gordenin, Ph.D., of the Chromosome Stability Group. (Photo courtesy of Steve McCaw)

Andreas Larrea, Ph.D.
Larrea joined Kunkel's group in June 2008 and left in May 2010. He is currently a field applications specialist with Pacific Biosciences. (Photo courtesy of Steve McCaw)

Scott Lujan, Ph.D.
Second author Scott Lujan is studying the two other major replicative DNA polymerases for insights into DNA repair mechanisms linked to the development of cancer. (Photo courtesy of Steve McCaw)

New research performed by NIEHS scientists may provide the answer to a decades-old question about the fundamentals of DNA replication.

The team, led by Thomas Kunkel, Ph.D. (http://www.niehs.nih.gov/research/atniehs/labs/lmg/dnarf/index.cfm), chief of the NIEHS Laboratory of Structural Biology and leader of the DNA Replication Fidelity Group, published its findings (http://www.ncbi.nlm.nih.gov/pubmed/20876092) Exit NIEHS in The Proceedings of the National Academy of Sciences U S A. The work is the latest in a series of studies that determine the proteins responsible for replicating the different DNA strands across the genome.

Kunkel noted that the two strands of DNA are replicated asymmetrically, with the leading strand replicated continuously, and the lagging strand replicated slightly thereafter and discontinuously in small stretches. "The human genome encodes 15 different DNA polymerases, three of which are responsible for the vast majority of nuclear DNA replication," he said. "The respective workload for these three polymerases has been debated for many years."

Flags on the genome

The research group used yeast to search for the mechanism of leading and lagging strand synthesis for two major reasons. First, the various locations where replication begins, called origins, have been determined in yeast, and identification of the leading and lagging strand can be predicted. Second, yeast is easy to grow and manipulate genetically.

The authors used yeast mutants containing error-prone versions of the three polymerases that cause very specific mutations. The location of the mutations acted as flags to determine the stretch of DNA that the polymerase replicated. In addition, the mutant strains contained a reporter gene in different orientations near an origin of replication. Thus, the spectrum of the mutations in the reporter gene determines whether the polymerase error occurred on the leading or lagging strand.

These results suggested distinct roles of the major replicative polymerases, epsilon and delta. Polymerase  epsilon was proposed to replicate the leading strand, while Polymerase delta (Pol delta) was proposed to replicate the lagging strand.

Last generation questions answered by next generation sequencing

Despite the elegance and clarity of the initial studies, the interpretations were based on investigations of a single origin of replication of only one of 16 yeast chromosomes, prompting the analogy of looking only under a lamp post. So, what happens in the rest of the genome?

The advent of high throughput sequencing created the opportunity to cast a light on the entire genome. Using strains that eliminate the repair of polymerase mistakes, enough mutations were present to track where the error-prone Pol delta had synthesized DNA. Near each origin, where the identification of leading and lagging strand is clear, Pol delta replicated only the lagging strand. However, closer to the middle of the origins where the two replication forks meet and either could be the lagging strand, Pol delta replicated either strand depending on the length of leading strand synthesis from each origin. As a result, this study demonstrated the probability that any region of the genome occurred by lagging strand synthesis.

These techniques may be expanded to human cells, and Kunkel predicts further insights. "Efforts are underway to study the two other major replicative DNA polymerases, and to study how replication errors are corrected, because failure to do so contributes to cancer," he explained.

Kunkel added, "Deep sequencing technology should provide a better understanding of how environmental stresses destabilize the genome, and may help to identify informative mutations present in cancer genomes. One can imagine a large number of applications because the whole genome is now the dosimeter to monitor cellular responses to environmental stress."

Andreas Larrea, Ph.D., a former Intramural Research Training Award (IRTA) postdoctoral fellow in Kunkel's group (http://www.niehs.nih.gov/research/atniehs/labs/lmg/dnarf/staff.cfm), was first author on the paper. Kunkel explained that the second author on the study, IRTA Fellow Scott Lujan, Ph.D., is continuing this line of research with other members of the Laboratory of Molecular Genetics (LMG).

Citation: Larrea AA, Lujan SA, Nick McElhinny SA, Mieczkowski PA, Resnick MA, Gordenin DA, Kunkel TA. (http://www.ncbi.nlm.nih.gov/pubmed/20876092) Exit NIEHS 2010. Genome-wide model for the normal eukaryotic DNA replication fork. Proc Natl Acad Sci U S A 107(41):17674-17679.

(Jeffrey Stumpf, Ph.D., is a postdoctoral fellow in the NIEHS Laboratory of Molecular Genetics Mitochondrial DNA Replication Group.)



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