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Research Offers New Insight into DNA Synthesis

By Bono Sen
April 2010

Stephanie Nick McElhinny, Ph.D.
During her fellowship at NIEHS, Nick McElhinny was a member of the DNA Replication Fidelity Group. (Photo courtesy of Steve McCaw)

Thomas Kunkel, Ph.D.
Kunkel is head of the Laboratory of Molecular Genetics DNA Replication Fidelity Group, chief of the Laboratory of Structural Biology, and director of the Environmental Biology Program at NIEHS. (Photo courtesy of Steve McCaw)

A new NIEHS-funded study challenges current thinking about ribonucleotide incorporation into DNA by yeast DNA polymerases and may offer new insights into mechanisms by which the human genome is stabilized and, conversely, may be destabilized. Published online in the Proceedings of the National Academies of Sciences, the study was led by NIEHS Principal Investigator Thomas Kunkel, Ph.D., (http://www.niehs.nih.gov/research/atniehs/labs/lmg/dnarf/index.cfm) and first authored by a former postdoctoral fellow in his group, Stephanie Nick McElhinny, Ph.D.

The study (http://www.ncbi.nlm.nih.gov/pubmed/20194773)Exit NIEHS suggests that ribonucleoside triphosphates (rNTPs) are incorporated into DNA as ribonucleoside monophosphates, or rNMPs, in much higher amounts than may have previously been appreciated. One likely reason for this is that the concentrations of the rNTPs needed for a wide variety of functions in yeast and mammalian cells greatly exceed those of the deoxyribonucleotides (dNTPs) needed for DNA replication and repair.

Most DNA polymerases are thought to efficiently prevent incorporation of rNTPs during DNA synthesis. To test this idea with polymerases that replicate the nuclear genome, these researchers determined the selectivity with which yeast DNA polymerases alpha (Pol α), delta (Pol δ) and epsilon (Pol ε) incorporate the monophosphates dNMPs and rNMPs during DNA synthesis in vitro. They found that, while all three polymerases prefer to incorporate dNMPs, rNMP incorporation into during DNA was surprisingly high.

Kunkel and his colleagues further showed that rNMP incorporation along a DNA template varied widely as a function of the polymerase, the identity of the base and the sequence context. Based on the estimated amount of DNA copied by the three polymerases during replication in vivo, the authors estimated that more than 13,000 rNMPs could be incorporated into DNA during each replication cycle in yeast.

The human genome is 500 times larger, so if human polymerases behave similarly - something that is currently unknown - it is possible that several million rNMPs could be introduced into the human genome during each cell division, potentially making rNMPs the most common of all noncanonical nucleotides introduced into the human genome.

The authors went on to consider the implications of abundant rNTP incorporation into DNA. They considered how rNMPs in DNA might be repaired, as well as the possibility that rNMPs in DNA might be tolerated reasonably well. However, given that ribonucleotides in DNA promote changes in helix geometry, unrepaired rNMPs in DNA may not be totally harmless, for example potentially resulting in mutations.

The researchers also considered the possibility that rNMPs in DNA might have beneficial consequences. They speculated that the presence of helix distorting rNMPs in DNA may serve signaling functions, perhaps for mismatch repair, nucleosome loading, chromatin remodeling, and gene silencing.

Coauthors on the study included scientists from the NIEHS Laboratory of Molecular Genetics, Umeå University in Sweden, and Washington University in St. Louis.

Citation: Nick McElhinny SA, Watts BE, Kumar D, Watt DL, Lundström EB, Burgers PM, Johansson E, Chabes A, Kunkel TA. (http://www.ncbi.nlm.nih.gov/pubmed/20194773)Exit NIEHS 2010. Abundant ribonucleotide incorporation into DNA by yeast replicative polymerases. Proc Natl Acad Sci U S A Mar 1. [Epub ahead of print]

(Bono Sen, Ph.D., is the science education and outreach program manager for the NIEHS journal Environmental Health Perspectives.)



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