Environmental Factor, November 2007, National Institute of Environmental Health Sciences
Intramural Papers of the Month
By Robin Arnette
- Identification of a New Base Excision Repair Cofactor
- A Novel Mechanism for Integrating Inositol Phosphate Signaling Pathways
- Hydrophobicity is Necessary in nAChR Signaling
- The Role of Accessory Proteins in Translesion DNA Synthesis
Identification of a New Base Excision Repair Cofactor
An international team of researchers jointly funded by NIEHS and the Japanese government have reported that high-mobility group box 1 (HMGB1)-a nuclear non-histone chromosomal protein that transiently introduces bends into linear DNA-is a cofactor involved in mammalian base excision repair (BER).
When cells are exposed to genotoxic chemicals such as methylating agents, deoxyribose phosphate (dRP) often appears as a cytoxic lesion at the 5' side of a DNA gap (5'-dRP). Removal of the 5'-dRP BER intermediate, primarily performed by Pol ?, allows proper BER function to continue. However, when this Pol ? activity is deficient, other BER cofactors must be able to remove the 5'-dRP. To determine which mammalian proteins are capable of interacting with 5'-dRP BER intermediates and removing the 5'-dRP group, the team preformed experiments with a Pol ? null mouse embryonic fibroblast cell extract. A protein and DNA complex capable of forming a Schiff base with the 5'-dRP intermediate was isolated and then the protein in the complex was identified by mass spectrometry analysis.
The analysis revealed that HMGB1 was the major BER intermediate interactive protein in the extract. Subsequent experiments using photoaffinity labeling, DNA/protein-binding assays, enzymatic assays, and immunofluorescence with GFP-tagged HMGB1 determined that HMGB1 in the cell both stimulates base excision repair and interacts with BER-related enzymes. HMGB1 appears to serve as a platform upon which the enzymatic transactions of DNA repair occur more efficiently.
This report is the first to identify the chromosomal protein HMGB1 as a base excision repair accessory factor.
Citation: Prasad R, Liu Y, Deterding LJ, Poltoratsky VP, Kedar PS, Horton JK, Kanno SI, Asagoshi K, Hou EW, Khodyreva SN, Lavrik OI, Tomer KB, Yasui A, Wilson SH. 2007. HMGB1 is a cofactor in mammalian base excision repair. Mol Cell. 27(5):829-841.
A Novel Mechanism for Integrating Inositol Phosphate Signaling Pathways
Scientists from NIEHS, Novartis Research Foundation and Virginia Tech have published a report in the Journal of Biological Chemistry demonstrating that a human inositol phosphate kinase (hITPK1) catalyzes phosphate exchange between two inositol phosphates from separate metabolic branches of this cell-signaling family. NIEHS and the U.S. Department of Energy funded the study.
Inositol 1,3,4?trisphosphate [Ins(1,3,4)P3] is a by-product of a signaling cascade that elevates cytoplasmic calcium. [Ca2+]. Ins(1,3,4)P3 exists in a metabolic pathway that is separate from inositol 1,3,4,5,6?pentakisphosphate [Ins(1,3,4,5,6)P5], which ITPK1 dephosphorylates to inositol 3,4,5,6-tetrakisphosphate [Ins(3,4,5,6)P4],an inhibitor of plasma membrane chloride channels. This study shows that hITPK1 functionally interconnects these two signaling pathways via an unprecedented "intersubstrate phosphate transfer" reaction: Ins(1,3,4)P3 accepts the 1-phosphate from Ins(1,3,4,5,6)P5, yielding Ins(3,4,5,6)P4. ITPK1 homologues from soybean and Entamoeba histolytica are not active phosphotranferases. To explain why the homologues are not active, the authors obtained structural data by crystallographic analysis, and then performed site-directed mutagenesis. They concluded that phosphotransferase activity requires amino-acid residues in the hITPK1 active site that are absent in plant and protozoan homologues.
These studies show how the catalytic activity of hITPK1 impacts regulation of diverse mammalian cellular functions such as fluid secretion, insulin secretion and neurotransmission.
Citation: Chamberlain PP, Qian X, Stiles AR, Cho J, Jones DH, Lesley SA, Grabau EA, Shears SB, Spraggon G. 2007. Integration of inositol phosphate signaling pathways via human ITPK1 J Biol Chem. 282(38):28117-28125.
Hydrophobicity is Necessary in nAChR Signaling
In a study funded by the NIH Intramural Research Program and published in the October issue of Molecular Pharmacology, NIEHS investigators from the Laboratory of Neurobiology and the Scientific Computing Laboratory reported that hydrophobic interactions are essential for apolipoprotein E (apoE) inhibition of acetylcholine responses mediated by the neuronal nicotinic acetylcholine receptor (nAChR). Drugs that act on nAChRs are promising therapeutic targets for many neurological disorders such as Alzheimer's disease, Parkinson's disease, epilepsy and schizophrenia.
Previous studies had determined that peptides derived from apoE inhibited native and recombinant homomeric alpha-7 subunits (?7) of nAChRs in Xenopus laevis oocytes, but the team wanted to study the specific interaction responsible for channel inhibition. The researchers used binding studies, site-directed mutagenesis of ?7 nAChRs and mutated apoE peptides to characterize the binding interaction. They created multiple ?7 nAChR mutants including a tryptophan to alanine mutant (?7-W55A), and also mutated apoE141-148-the peptide that inhibited nAChR function-by substituting two positively charged lysines with leucines (apoE141-1482K/2L) or glutamic acid (apoE141-1482K/2E).
In assays, the ?7-W55A mutant demonstrated diminished inhibition by apoE peptides of the acetylcholine-activated ?7 nAChR. Computer modeling of apoE peptide docking to the ?7 nAChR confirmed the functional data. The hydrophobic interactions generated between apoE141-148 and the ?7 nAChR interface are necessary for peptide inhibition of nAChR signaling.
Citation: Gay EA, Bienstock RJ, Lamb PW, Yakel JL. 2007. Structural determinates for apolipoprotein E-derived peptide interaction with the alpha 7 nicotinic acetylcholine receptor. Mol Pharmacol. 72(4):838-849.
The Role of Accessory Proteins in Translesion DNA Synthesis
In a study published in the journal Biochemistry, researchers from NIEHS and Washington University School of Medicine in St. Louis reported on the effects of replication accessory proteins on translesion synthesis (TLS) by DNA polymerase eta (pol eta) in the budding yeast Saccharomyces cerevisiae. The study was funded by the NIEHS Intramural Research Program and the NIH.
TLS allows organisms to bypass lesions that block DNA synthesis by replicative polymerases. Human and yeast cells lacking pol eta display increased mutagenesis following ultraviolet light (UV) exposure, indicating that pol eta suppresses mutagenesis caused by UV-induced DNA damage, and humans lacking pol eta are highly susceptible to skin cancer. Pol eta is known to perform low-fidelity bypass of the UV light induced cis-syn thymine-thymine dimer (TT dimer), and the team asked whether other proteins could improve the efficiency and/or the accuracy of this bypass.
The investigators measured the efficiency and fidelity of TT dimer bypass by pol eta in the absence or presence of proteins involved in DNA replication: RPA (single stranded DNA binding protein), PCNA (the eukaryotic sliding clamp) and RFC (the clamp loader complex). The results demonstrated that these three accessory proteins have at most subtle effects on TLS efficiency and fidelity, indicating that the pol eta itself plays a dominant role in the TLS-dependent suppression of UV light-induced mutagenesis and carcinogenesis in humans.
Citation: McCulloch SD, Wood A, Garg P, Burgers PMJ, Kunkel TA. 2007. Effects of accessory proteins on the bypass of a cis-syn thymine-thymine dimer by Saccharomyces cerevisiae DNA polymerase eta. Biochemistry. 46(30):8888-8896.