Human glutaredoxin (GRx), also known as thioltransferase, is a 12 kDa thiol-disulfide oxidoreductase that highly selective for reduction of glutathione-containing mixed disulfides. The apparent pKa the active site Cys22 residue approximately 3.5. Previously we observed catalytic enhancement by could be ascribed fully to difference between its thiol moiety and product thiol, each acting leaving group in enzymatic nonenzymatic reactions, respectively [Srinivasan et al. (1997), Biochemistry 36,...

Despite the nontemplating nature of abasic site, dAMP is often preferentially inserted opposite lesion, a phenomenon commonly referred to as "A-rule". We have evaluated molecular mechanism accounting for this unique behavior using thorough kinetic approach evaluate polymerization efficiency during translesion DNA replication. Using bacteriophage T4 polymerase, we measured insertion series modified nucleotides and demonstrated that increasing size nucleobase does not correlate with increased...

The kinetic properties of an adenine DNA methyltransferase involved in cell cycle regulation Caulobacter crescentus have been elucidated by using defined unmethylated or hemimethylated (DNA HM ) substrates. Catalytic efficiency is significantly enhanced with a substrate. Biphasic behavior during methyl incorporation observed when substrates are used, indicating that step after chemistry limits enzyme turnover and most likely the release from methylated product. thermally inactivated at 30°C...

This report describes the design and application of several distinct gold-containing indoles as anticancer agents. When used individually, all gold-bearing compounds display cytostatic effects against leukemia adherent cancer cell lines. However, two show unique behavior by increasing cytotoxic clinically relevant levels ionizing radiation. Quantifying amount DNA damage demonstrates that each gold-indole enhances apoptosis inhibiting repair. Both Au(I)-indoles were tested for inhibitory...

Telomerase, the end-replication enzyme, is reactivated in malignant cancers to drive cellular immortality. While this distinction makes telomerase an attractive target for anti-cancer therapies, most approaches inhibiting its activity have been clinically ineffective. As opposed telomerase, we use selectively promote cytotoxicity cancer cells. We show that several nucleotide analogs, including 5-fluoro-2'-deoxyuridine (5-FdU) triphosphate, are effectively incorporated by into a telomere DNA...

Translesion DNA synthesis (TLS) by specialized polymerases (Pols) is a conserved mechanism for tolerating replication blocking lesions. The actions of TLS Pols are managed in part ring-shaped sliding clamp proteins. In addition to catalyzing TLS, altered expression impedes cellular growth. goal this study was define the relationship between physiological function Escherichia coli Pol IV and its ability impede growth when overproduced. To end, 13 novel mutants were identified that failed...

Steady-state and pre-steady-state rates of ATP hydrolysis by the 44/62 accessory protein were determined to elucidate role in bacteriophage T4 holoenzyme complex formation. ATPase measurements under various combinations 45 protein, DNA substrate, exo- polymerase indicate that although synergistically hydrolyzes presence activity is diminished substantially upon formation complex. The decrease primarily kcat while K(m) for changed unsubstantially combinations. Data suggest rate addition...

To further elucidate the mechanism and dynamics of bacteriophage T4 holoenzyme formation, a mutant polymerase in which last six carboxyl-terminal amino acids are deleted, was constructed, overexpressed, purified to homogeneity. The polymerase, designated ΔC6 exo − , is identical wild-type with respect k cat pol dissociation constants for nucleotide DNA substrate. However, unlike unable interact 45 protein form stable holoenzyme. A synthetic polypeptide corresponding carboxyl terminus tested...

It is widely accepted that the dynamic behavior of DNA polymerases during translesion synthesis dependent upon nature lesion and incoming dNTP destined to be complementary partner. We previously demonstrated 5-nitro-1-indolyl-2'-deoxyribose-5'-triphosphate, a nonnatural nucleobase possessing enhanced base-stacking abilities, can selectively incorporated opposite an abasic site (Reineks, E. Z., Berdis, A. J. (2004) Biochemistry 43, 393−404.). While enhancement in insertion presumably...

Translesion DNA synthesis, the ability of a polymerase to misinsert nucleotide opposite damaged template, represents common route toward mutagenesis and possibly disease development. To further define mechanism this promutagenic process, we synthesized tested enzymatic incorporation two isosteric 5-substituted indolyl-2'deoxyriboside triphosphates an abasic site. The catalytic efficiency for 5-cyclohexene-indole derivative site is 75-fold greater than that 5-cyclohexyl-indole derivative....

Abstract Fundamental study of enzymatic nucleoside transport suffers for lack optical probes that can be tracked noninvasively. Nucleoside transporters are integral membrane glycoproteins mediate the salvage nucleosides and their passage across cell membranes. The substrate recognition site is deoxyribose sugar, often with little distinction among nucleobases. Reported here analogues in which emissive, cyclometalated iridium(III) complexes “clicked” to C‐1 place canonical resulting show...

Most biological organisms rely upon a DNA polymerase holoenzyme for processive replication. The bacteriophage T4 is composed of the enzyme and clamp protein (the 45 protein), which functions as processivity factor by strengthening interaction between holoenzyme. must be loaded onto loader ATPase complex 44/62 complex). In this paper, order events leading to formation investigated using combination rapid-quench stopped-flow fluorescence spectroscopy kinetic methods. A strand displacement...

The effect of metal ion substitution on the dynamics translesion DNA synthesis catalyzed by bacteriophage T4 polymerase was quantitatively evaluated through steady-state and transient kinetic techniques. Substitution Mn(2+) for Mg(2+) enhances rate dNMP misinsertion opposite an abasic site 11-34-fold. At molecular level, enhancement in reflects a substantial increase conformational change preceding phosphoryl transfer all dNTPs that were tested. This is best illustrated biphasic...

Acute lymphoblastic leukemia (ALL) is the most common type of childhood cancer, presenting with approximately 5,000 new cases each year in United States. An interesting enzyme implicated this disease terminal deoxynucleotidyl transferase (TdT), a specialized DNA polymerase involved V(D)J recombination. TdT an excellent biomarker for ALL as it overexpressed ~90% patients, and these higher levels correlate poor prognosis. These collective features make attractive target to design selective...

Temozolomide is a DNA-alkylating agent used to treat brain tumors, but resistance this drug common. In study, we provide evidence that efficacious responses can be heightened significantly by coadministration of an artificial nucleoside (5-nitroindolyl-2'-deoxyriboside, 5-NIdR) efficiently and selectively inhibits the replication DNA lesions generated temozolomide. Conversion compound corresponding triphosphate, 5-nitroindolyl-2'-deoxyriboside in vivo creates potent inhibitor several human...

Telomerase, a unique reverse transcriptase that specifically extends the ends of linear chromosomes, is up-regulated in vast majority cancer cells. Here, we show an indole nucleotide analog, 5-methylcarboxyl-indolyl-2′-deoxyriboside 5′-triphosphate (5-MeCITP), functions as inhibitor telomerase activity. The crystal structure 5-MeCITP bound to Tribolium castaneum reveals atypical interaction, which nucleobase flipped active site. In this orientation, methoxy group out canonical site interact...