Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.16.2 (PCP)
 3,761 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Lysine 318 in the conserved sequence SXXXGXGKS of bacteriophage T7 gene 4A' protein was mutated to an alanine to understand the effect of this substitution on the helicase and primase activities. The dTTPase activity of 4A'/K318A mutant protein was much lower than that of 4A', and both Km and kcat values were affected. The Km of the mutant protein was 3-5-fold higher, and the kcat was about 100-fold lower, than that of 4A'. The mutation did not affect the ability of 4A'/K318A to assemble into hexamers or bind DNA in the presence of MgdTTP. Interestingly, the mutant protein does not bind DNA in the presence of MgdTMP-PCP. The reduced dTTPase activity, however, decreased the helicase activity of the mutant protein to an undetectable level, whereas its primase activity was only 1.5-2.5-fold lower. When 4A'/K318A mutant protein was mixed with 4A', heterooligomers were formed and the helicase and the DNA-dependent dTTPase activities of 4A' were inhibited, but the DNA-independent activity actually increased. The extent of decrease in activities upon heterooligomer formation depended both on the length of time 4A' and 4A'/K318A proteins were incubated and on the concentration of the mutant protein. In addition, the decrease in the dTTPase activity was observed only when the two proteins were incubated in the absence of MgdTTP and DNA, conditions under which both proteins form unstable hexamers. Even though 4A'/K318A does not bind a 30-mer DNA in the presence of MgdTMP-PCP, heterooligomers were capable of binding DNA with the same stoichiometry as 4A'. Protein-DNA cross-linking experiments with (dT)30 and poly(5-BrdU) showed that DNA interacts with five and perhaps all six subunits of 4A'. Therefore, unless heterooligomer restores the ability of the mutant protein to bind DNA in the presence of MgdTMP-PCP, these results suggest that the DNA can bind 4A' by interacting with a few subunits. However, a fully active hexamer is required for both the helicase and the single-stranded M13 DNA-dependent dTTPase activities.
 ...
PMID:The K318A mutant of bacteriophage T7 DNA primase-helicase protein is deficient in helicase but not primase activity and inhibits primase-helicase protein wild-type activities by heterooligomer formation. 801 49

Protein-DNA interactions of bacteriophage T7 DNA primase/helicase protein 4A' with small synthetic oligodeoxynucleotides were investigated using a 20-base-paired hairpin duplex, and 10-, 30-, and 60-base-long single-stranded DNA. The effect of nucleotide cofactors on DNA binding was examined using membrane binding assays which showed that 4A' binds DNA optimally only in the presence of MgdTMP-PCP, the nonhydrolyzable analog of dTTP. About 20% of single-stranded DNA binding was observed in the presence of MgdTDP, but none was detectable in the absence of nucleotides. Native polyacrylamide gel electrophoresis showed that the DNAs bind predominantly to the hexameric form of 4A'. Larger oligomers of 4A' can bind DNA, but no DNA binding was observed to species smaller than the hexamer. Quantitative equilibrium binding studies at increasing 4A' concentrations and at increasing DNA concentrations showed tight binding of one 10-mer or 30-mer per hexamer. The 4A' hexamer can bind a second strand of DNA, but with a 50-fold weaker affinity than the first strand. The 60-mer showed tight binding to two 4A' hexamers, suggesting that a hexamer may interact with only 30-40 bases of single-stranded DNA. This was corroborated by nuclease protection experiments where the smallest length of DNA protected by 4A' or 4B protein was found to be about 30 bases. Equilibrium binding studies and competitive DNA binding data are consistent with a weaker affinity of 4A' for the duplex DNA. Only 20-25% of duplex DNA binding was observed at increasing 4A' protein in the presence of MgdTMP-PCP. About four duplex DNAs can bind each 4A' hexamer at increasing DNA concentrations, but their weaker binding was evident from their facile dissociation from 4A' in the presence of competing single-stranded DNA.
 ...
PMID:Interactions of bacteriophage T7 DNA primase/helicase protein with single-stranded and double-stranded DNAs. 824 Nov 39

Many helicases assemble into ring-shaped hexamers and bind DNA in their central channel. This raises the question as to how the DNA gets into the central channel to form a topologically linked complex. We have used the presteady-state stopped-flow kinetic method and protein fluorescence changes to investigate the mechanism of single-stranded DNA (ssDNA) binding to the bacteriophage T7 helicase-primase, gp4A'. We have found that the kinetics of 30-mer ssDNA binding to a preformed gp4A' hexamer in the presence of both Mg-dTMP-PCP and Mg-dTTP are similar, indicating that Mg-dTTP binding is sufficient and hydrolysis is not necessary for efficient DNA binding. Multiple transient changes in gp4A' fluorescence revealed a four-step mechanism for DNA binding with Mg-dTTP. These transient changes were analyzed by global fitting and kinetic simulation to determine the intrinsic rate constants of this four-step mechanism. The initial steps, including the bimolecular encounter of the DNA with the helicase and a subsequent conformational change, were fast. We propose that these initial steps of DNA binding occur at a readily accessible site, which is likely to be on the outside of the hexamer ring. The binding of the 30-mer ssDNA at this loading site is followed by slower conformational changes that allow the DNA to transit into the central channel of gp4A' via a ring-opening or threading pathway.
 ...
PMID:DNA binding in the central channel of bacteriophage T7 helicase-primase is a multistep process. Nucleotide hydrolysis is not required. 1082 54

c-jun-N-Terminal kinase 3alpha1 (JNK3alpha1) is a mitogen-activated protein (MAP) kinase family member expressed primarily in the brain that phosphorylates protein transcription factors including c-jun and activating transcription factor 2 (ATF2) upon activation by a variety of stress-based stimuli. In this study, the kinetic mechanism for JNK3alpha1 was determined via initial velocity patterns in the presence and absence of both ATP and ATF2 competitive inhibitors. Peptide inhibitors from both ATF2 (peptide 1) and JNK-interacting protein 1 (JIP-1) (peptide 3), derived from the homologous delta-domain JNK docking sequence, inhibited JNK3alpha1 activity in a competitive fashion versus ATF2 while being pure noncompetitive toward ATP. In contrast, peptides derived from the phosphoacceptor activation domain on ATF2 (peptides 4 and 5) were recognized neither as substrates nor as inhibitors of JNK3alpha1. AMP-PCP and compound 6, a small molecule analinopyrimidine, exhibited pure noncompetitive inhibition versus ATF2 and competitive inhibition versus ATP. Peptide inhibitors based on the delta-domain sites of JIP ( 3) and ATF2 ( 1) were not recognized by p38, also of the MAPK family, which may give insight into finding more selective inhibitors for the JNK family of kinases. Collectively these data showed that JNK3alpha1 proceeded by a random sequential kinetic mechanism and that the ATP and ATF2 substrate sites were non-interacting. Moreover, these results established the 11-mer JIP peptide ( 3) as a potent ( K i = 25 +/- 6 nM) competitive inhibitor versus ATF2 in JNK3alpha1.
 ...
PMID:Kinetic mechanism and inhibitor characterization for c-jun-N-terminal kinase 3alpha1. 1826 48

Certain phenoxyl radicals can attach covalently to the C8-site of 2'-deoxyguanosine (dG) to afford oxygen-linked C8-dG adducts. Such O-linked adducts can be chemically synthesized through a nucleophilic displacement reaction between a phenolate and a suitably protected 8-Br-dG derivative. This permits the generation of model O-linked C8-dG adducts on scales suitable for insertion into oligonucleotide substrates using solid-phase DNA synthesis. Variation of the C8-aryl moiety provides an opportunity to derive structure-activity relationships on adduct conformation in duplex DNA and replication bypass by DNA polymerases. In the current study, the influence of chlorine C8-dG functionalization on in vitro DNA replication by Klenow fragment exo(-) (Kf(-)) and the Y-family polymerase (Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4)) has been determined. Model O-linked C8-dG adducts derived from the pentachlorophenoxyl radical ([PCP]G) and 2,4,6-trichlorophenoxyl radical ([TCP]G) were inserted into the reiterated G3-position of the NarI sequence (12-mer, NarI(12); and 22-mer, NarI(22)), which is a known hotspot for frameshift mutations mediated by N-linked polycyclic C8-dG adducts in bacterial mutagenesis. Within the NarI(12) duplex, the unsubstituted C8-phenoxy-dG ([PhO]G) adduct adopts a minimally perturbed B-form helix. Chlorination of [PhO]G to afford [PCP]G does not significantly change the adduct conformation within the NarI(12) duplex, as predicted by molecular dynamics simulations. However, when using NarI(22) for DNA synthesis in vitro, the chlorinated [PCP]G and [TCP]G lesions significantly block DNA replication by Kf(-) and Dpo4, whereas [PhO]G is readily bypassed. These findings highlight the impact that chlorine substituents impart to bulky C8-dG lesions.
 ...
PMID:Chlorine functionalization of a model phenolic C8-guanine adduct increases conformational rigidity and blocks extension by a Y-family DNA polymerase. 2600 22