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August 2011

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Ribonucleotide incorporation into DNA study honored by JBC

By Jeffrey Stumpf
August 2011

Samuel  Wilson, M.D.

In his roles as a leader and researcher at NIEHS, Wilson has long championed research on the role of DNA pol beta in DNA repair and replication. As principal investigator on the study, Wilson led a multifaceted approach toward understanding the enzyme that involved a collaborative effort between members of his own group -William Beard, Ph.D., Vinod Batra, Ph.D., Cavanaugh, and David Shock - and members of the LSB Computational Chemistry and Molecular Modeling Group - head Lee Pedersen, Ph.D., and Lalith Perera, Ph.D. (Photo courtesy of Steve McCaw)

Nisha Cavanaugh, Ph.D.

Cavanaugh was the first author on the JBC paper of the week. Her research was also honored by NIH with a Fellows Award for Research Excellence. (Photo courtesy of Steve McCaw)

Research from the NIEHS Laboratory of Structural Biology (LSB) provides the latest chapter in the story of a rapidly growing field that investigates the role of ribonucleotide incorporation into DNA and how these basic processes may affect the health of an organism and its response to endogenous cellular metabolism or exposure to the environment.

In July, the Journal of Biological Chemistry (JBC) honored a new study( Exit NIEHS by LSB investigators on the biochemical and structural characterization of ribonucleotide incorporation by human DNA polymerase beta (pol beta) by naming it Paper of the Week. JBC associate editors and editorial board members bestow this honor on the top one percent of papers reviewed in terms of significance and overall importance, according to the JBC website.

A fundamental role of DNA polymerases is to insert the correct deoxyribonucleoside triphosphate (dNTP) opposite a DNA template, discriminating against an incorrect base or ribonucleoside triphosphate (rNTP). However, members of the LSB DNA Repair and Nucleic Acid Enzymology Group, headed by Samuel Wilson, M.D., are the latest to show that polymerases can introduce ribonucleotides frequently during DNA replication, as much as two percent of the time for pol beta. Although the biological consequences are unknown, lead author Nisha Cavanaugh, Ph.D., a postdoctoral fellow in the group, said she suspects that limiting rNTP incorporation may be important for normal cell survival.

“Because ribonucleotides are susceptible to hydrolysis, their presence in the genome will result in DNA strand breaks and genome instability,” noted Cavanaugh. “Additionally, rNTP incorporation may disrupt nucleic acid binding proteins, such as transcription factors, that rely on specific sequences or conformations.”

Potential clinical implications

Ribonucleotides vastly outnumber dNTPs in growing cells, allowing for higher probability of rNTP incorporation into DNA. While the majority of the DNA replication field focuses on the study of incorporating the correct base, Wilson points out that sugar-modified nucleosides, such as AZT and araC, are used in the clinic for development of cancer therapies.

“Many aspects toward understanding mechanistic features of the cytotoxic activity of these compounds are poorly understood, and this field represents a really fascinating area for future investigation,” predicted Wilson.

Structural and kinetic studies of nucleotide incorporation by polymerases are key to this investigation. “By understanding how DNA polymerases discriminate against sugar or base analogs, we can develop better therapeutic nucleotide analogs that could target specific DNA polymerases,” added Cavanaugh.

Good fences make good polymerases

Pol beta fills short gaps that result from the process that repairs simple DNA lesions, called base excision repair. Similar to other DNA polymerases, pol beta discriminates against rNTP incorporation using an important tyrosine residue 271 (Tyr-271), whose side chain and peptide backbone carbonyl forms a steric fence. Different polymerases contain different steric gate residues, but the importance of this variation on rNTP incorporation is unknown.

The researchers purified mutant pol beta where tyrosine was substituted with alanine (Y271A), thus eliminating the majority of the side chain. By determining the three-dimensional structure of the Y271A mutant bound to DNA and the incorporated rNTP, the authors realized that both the tyrosine backbone and the side chain inhibit rNTP incorporation, prompting the comparison to a steric fence rather than just a gate.

Landscaping aside, at least two other factors influence rNTP incorporation. Structural data suggest a hydrogen bond between the side chain of Tyr-271 and the primer terminus that may provide a geometry that discriminates against ribonucleotides. Also, kinetic analyses show that substituting manganese for magnesium as the chelating metal ion increases rNTP binding affinity 200-fold. However, Cavanaugh asserts that adjustments of the reactive atoms in the active site that deter ribonucleotide insertion are subtle.

“The crystallographic structure of pol beta indicates that the ribonucleotide is well accommodated,” observed Cavanaugh. “This data contributes to the mechanism that explains why pol Beta inserts ribonucleotides much more often than deoxyribonucleotides with the wrong base.”

Citation: Cavanaugh NA, Beard WA, Batra VK, Perera L, Pedersen LG, Wilson SH.( Exit NIEHS 2011. Molecular insights into DNA polymerase deterrents for ribonucleotide insertion. J Biol Chem; doi/10.1074/jbc.M111.253401 [Online 6 July 2011].

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

Paper expands NIEHS interest in rNTPs incorporation into DNA

Recent emerging interest by NIEHS researchers in ribonucleotide incorporation into DNA reflects the importance of this work to environmental health and disease. Although this subject has lacked sufficient investigation throughout the decades of mutagenesis research, the importance of DNA repair of ribonucleotides is now evident.

Leading the charge is LSB Chief Thomas Kunkel, Ph.D., whose lab uses biochemical and genetic tools to measure the effects of ribonucleotide incorporation into DNA. He notes that the disease relevance is already there. “RNase H2 initiates repair of ribonucleotides in DNA, and mutations in the genes encoding the three subunits of human RNase H2 are causally associated with Aicardi-Goutières syndrome,” stated Kunkel.

Mitochondrial DNA is not immune to ribonucleotide incorporation, which may cause mitochondrial disease. Chief of the Laboratory of Molecular Genetics William Copeland, Ph.D., is leading studies into how the mitochondrial DNA polymerase (pol gamma) discriminates against rNTPs. “We have already observed mitochondrial patients with mutant pol gamma that exhibits a loss of ribonucleotide discrimination,” said Copeland.

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