The inhibitor of κB kinase β (IKKβ) phosphorylates IκBα twice in a single binding event through a sequential mechanism.

Phosphorylation of Inhibitor of κB (IκB) proteins by IκB Kinase β (IKKβ) leads to IκB degradation and subsequent activation of nuclear factor κB transcription factors. Of particular interest is the IKKβ-catalyzed phosphorylation of IκBα residues Ser and Ser within a conserved destruction box motif. To investigate the catalytic mechanism of IKKβ, we performed pre-steady-state kinetic analysis of the phosphorylation of IκBα protein substrates catalyzed by constitutively active, human IKKβ.

Silicone breast implant rupture: a review.

Silicone breast implants have been in use for nearly 6 decades. In this time they have undergone significant changes in design and use. They have been subject to intense scrutiny with regard to safety and efficacy, including an almost 10 years moratorium on their use.

Backbone assignment of the binary complex of the full length Sulfolobus solfataricus DNA polymerase IV and DNA.

Sulfolobus solfataricus DNA polymerase IV (Dpo4), a model Y-family DNA polymerase, bypasses a wide range of DNA lesions in vitro and in vivo. In this paper, we report the backbone chemical shift assignments of the full length Dpo4 in its binary complex with a 14/14-mer DNA substrate. Upon DNA binding, several β-stranded regions in the isolated catalytic core and little finger/linker fragments of Dpo4 become more structured.

Structural and kinetic insights into binding and incorporation of L-nucleotide analogs by a Y-family DNA polymerase.

Considering that all natural nucleotides (D-dNTPs) and the building blocks (D-dNMPs) of DNA chains possess D-stereochemistry, DNA polymerases and reverse transcriptases (RTs) likely possess strongD-stereoselectivity by preferably binding and incorporating D-dNTPs over unnatural L-dNTPs during DNA synthesis. Surprisingly, a structural basis for the discrimination against L-dNTPs by DNA polymerases or RTs has not been established although L-deoxycytidine analogs (lamivudine and emtricitabine) and L-thymidine (telbivudine) have been widely used as antiviral drugs for years. Here we report seven high-resolution ternary crystal structures of a prototype Y-family DNA polymerase, DNA, and D-dCTP, D-dCDP, L-dCDP, or the diphosphates and triphosphates of lamivudine and emtricitabine.

Direct probing of solvent accessibility and mobility at the binding interface of polymerase (Dpo4)-DNA complex.

Water plays essential structural and dynamical roles in protein-DNA recognition through contributing to enthalpic or entropic stabilization of binding complex and by mediating intermolecular interactions and fluctuations for biological function. These interfacial water molecules are confined by the binding partners in nanospace, but in many cases they are highly mobile and exchange with outside bulk solution. Here, we report our studies of the interfacial water dynamics in the binary and ternary complexes of a polymerase (Dpo4) with DNA and an incoming nucleotide using a site-specific tryptophan probe with femtosecond resolution.

Identification of an unfolding intermediate for a DNA lesion bypass polymerase.

Sulfolobus solfataricus DNA Polymerase IV (Dpo4), a prototype Y-family DNA polymerase, has been well characterized biochemically and biophysically at 37 °C or lower temperatures. However, the physiological temperature of the hyperthermophile S. solfataricus is approximately 80 °C.

Presteady state kinetic investigation of the incorporation of anti-hepatitis B nucleotide analogues catalyzed by noncanonical human DNA polymerases.

Antiviral nucleoside analogues have been developed to inhibit the enzymatic activities of the hepatitis B virus (HBV) polymerase, thereby preventing the replication and production of HBV. However, the usage of these analogues can be limited by drug toxicity because the 5'-triphosphates of these nucleoside analogues (nucleotide analogues) are potential substrates for human DNA polymerases to incorporate into host DNA. Although they are poor substrates for human replicative DNA polymerases, it remains to be established whether these nucleotide analogues are substrates for the recently discovered human X- and Y-family DNA polymerases.

Backbone assignment of the little finger domain of a Y-family DNA polymerase.

Sulfolobus solfataricus DNA polymerase IV (Dpo4), a prototype Y-family DNA polymerase, contains a unique little finger domain besides a catalytic core. Here, we report the chemical shift assignments for the backbone nitrogens, α and β carbons, and amide protons of the little finger domain of Dpo4. This work and our published backbone assignment for the catalytic core provide the basis for investigating the conformational dynamics of Dpo4 during catalysis using solution NMR spectroscopy.

Pre-steady-state kinetic analysis of the incorporation of anti-HIV nucleotide analogs catalyzed by human X- and Y-family DNA polymerases.

Nucleoside reverse transcriptase inhibitors (NRTIs) are an important class of antiviral drugs used to manage infections by human immunodeficiency virus, which causes AIDS. Unfortunately, these drugs cause unwanted side effects, and the molecular basis of NRTI toxicity is not fully understood. Putative routes of NRTI toxicity include the inhibition of human nuclear and mitochondrial DNA polymerases.

Kinetic basis of sugar selection by a Y-family DNA polymerase from Sulfolobus solfataricus P2.

DNA polymerases use either a bulky active site residue or a backbone segment to select against ribonucleotides in order to faithfully replicate cellular genomes. Here, we demonstrated that an active site mutation (Y12A) within Sulfolobus solfataricus DNA polymerase IV (Dpo4) caused an average increase of 220-fold in matched ribonucleotide incorporation efficiency and an average decrease of 9-fold in correct deoxyribonucleotide incorporation efficiency, leading to an average reduction of 2000-fold in sugar selectivity. Thus, the bulky side chain of Tyr12 is important for both ribonucleotide discrimination and efficient deoxyribonucleotide incorporation.

Identification of critical residues for the tight binding of both correct and incorrect nucleotides to human DNA polymerase λ.

DNA polymerase λ (Pol λ) is a novel X-family DNA polymerase that shares 34% sequence identity with DNA polymerase β. Pre-steady-state kinetic studies have shown that the Pol λ-DNA complex binds both correct and incorrect nucleotides 130-fold tighter, on average, than the DNA polymerase β-DNA complex, although the base substitution fidelity of both polymerases is 10(-)(4) to 10(-5). To better understand Pol λ's tight nucleotide binding affinity, we created single-substitution and double-substitution mutants of Pol λ to disrupt the interactions between active-site residues and an incoming nucleotide or a template base.

Quantitative analysis of the efficiency and mutagenic spectra of abasic lesion bypass catalyzed by human Y-family DNA polymerases.

Higher eukaryotes encode various Y-family DNA polymerases to perform global DNA lesion bypass. To provide complete mutation spectra for abasic lesion bypass, we employed short oligonucleotide sequencing assays to determine the sequences of abasic lesion bypass products synthesized by human Y-family DNA polymerases eta (hPolη), iota (hPolι) and kappa (hPolκ). The fourth human Y-family DNA polymerase, Rev1, failed to generate full-length lesion bypass products after 3 h.

Backbone assignment of the catalytic core of a Y-family DNA polymerase.

Sulfolobus solfataricus DNA polymerase IV (Dpo4), a model Y-family DNA polymerase, bypasses DNA lesions. Here, we report the assignments for the backbone nitrogen, carbon, and amide proton NMR signals of Dpo4's catalytic core consisting of the finger, palm, and thumb domains. Our work provides the basis for further NMR spectroscopic studies of the interactions among Dpo4, DNA, and an incoming nucleotide.

Kinetic basis of nucleotide selection employed by a protein template-dependent DNA polymerase.

Rev1, a Y-family DNA polymerase, contributes to spontaneous and DNA damage-induced mutagenic events. In this paper, we have employed pre-steady-state kinetic methodology to establish a kinetic basis for nucleotide selection by human Rev1, a unique nucleotidyl transferase that uses a protein template-directed mechanism to preferentially instruct dCTP incorporation. This work demonstrated that the high incorporation efficiency of dCTP is dependent on both substrates: an incoming dCTP and a templating base dG.

A novel mechanism of sugar selection utilized by a human X-family DNA polymerase.

During DNA synthesis, most DNA polymerases and reverse transcriptases select against ribonucleotides via a steric clash between the ribose 2'-hydroxyl group and the bulky side chain of an active-site residue. In this study, we demonstrated that human DNA polymerase lambda used a novel sugar selection mechanism to discriminate against ribonucleotides, whereby the ribose 2'-hydroxyl group was excluded mostly by a backbone segment and slightly by the side chain of Y505. Such steric clash was further demonstrated to be dependent on the size and orientation of the substituent covalently attached at the ribonucleotide C2'-position.

Probing conformational changes of human DNA polymerase lambda using mass spectrometry-based protein footprinting.

Crystallographic studies of the C-terminal DNA polymerase-beta-like domain of full-length human DNA polymerase lambda (fPollambda) suggested that the catalytic cycle might not involve a large protein domain rearrangement as observed with several replicative DNA polymerases and DNA polymerase beta. To examine solution-phase protein conformational changes in fPollambda, which also contains a breast cancer susceptibility gene 1 C-terminal domain and a proline-rich domain at its N-terminus, we used a mass spectrometry-based protein footprinting approach. In parallel experiments, surface accessibility maps for Arg residues were compared for the free fPollambda versus the binary complex of enzyme*gapped DNA and the ternary complex of enzyme*gapped DNA*dNTP (2'-deoxynucleotide triphosphate).

Regulated renin release from 3T3-L1 adipocytes.

Whereas adipose tissue possesses a local renin-angiotensin system, the synthesis and regulated release of renin has not been addressed. To that end, we utilized differentiating 3T3-L1 cells and analyzed renin expression and secretion. Renin mRNA expression and protein enzymatic activity were not detectable in preadipocytes.

Mechanistic studies of the bypass of a bulky single-base lesion catalyzed by a Y-family DNA polymerase.

1-nitropyrene, the most abundant nitro polycyclic aromatic hydrocarbon in diesel emissions, has been found to react with DNA to form predominantly N-(deoxyguanosin-8-yl)-1-aminopyrene (dGAP). This bulky adduct has been shown to induce genetic mutations, which may implicate Y-family DNA polymerases in its bypass in vivo. To establish a kinetic mechanism for the bypass of such a prototype single-base lesion, we employed pre-steady-state kinetic methods to investigate individual nucleotide incorporations upstream, opposite, and downstream from a site-specifically placed dGAP lesion catalyzed by Sulfolobus solfataricus DNA polymerase IV (Dpo4), a model Y-family DNA polymerase.

Renin dynamics in adipose tissue: adipose tissue control of local renin concentrations.

The renin-angiotensin system (RAS) has been implicated in a variety of adipose tissue functions, including tissue growth, differentiation, metabolism, and inflammation. Although expression of all components necessary for a locally derived adipose tissue RAS has been demonstrated within adipose tissue, independence of local adipose RAS component concentrations from corresponding plasma RAS fluctuations has not been addressed. To analyze this, we varied in vivo rat plasma concentrations of two RAS components, renin and angiotensinogen (AGT), to determine the influence of their plasma concentrations on adipose and cardiac tissue levels in both perfused (plasma removed) and nonperfused samples.

Kinetic investigation of the inhibitory effect of gemcitabine on DNA polymerization catalyzed by human mitochondrial DNA polymerase.

Gemcitabine, 2'-deoxy-2', 2'-difluorocytidine (dFdC), is a drug approved for use against various solid tumors. Clinically, this moderately toxic nucleoside analog causes peripheral neuropathy, hematological dysfunction, and pulmonary toxicity in cancer patients. Although these side effects closely mimic symptoms of mitochondrial dysfunction, there is no direct evidence to show gemcitabine interferes with mitochondrial DNA replication catalyzed by human DNA polymerase gamma.

Single-turnover kinetic analysis of the mutagenic potential of 8-oxo-7,8-dihydro-2'-deoxyguanosine during gap-filling synthesis catalyzed by human DNA polymerases lambda and beta.

In the presence of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) damage, many DNA polymerases exhibit a dual coding potential which facilitates efficient incorporation of matched dCTP or mismatched dATP. This also holds true for the insertion of 8-oxodGTP opposite template bases dC and dA. Employing single-turnover kinetic methods, we examined human DNA polymerase beta and its novel X-family homolog, human DNA polymerase lambda, to determine which nucleotide and template base was preferred when encountering 8-oxodG and 8-oxodGTP, respectively.