Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:6.5.1.2 (DNA ligase)
 2,749 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human polynucleotide kinase (hPNK) is a putative DNA repair enzyme in the base excision repair pathway required for processing and rejoining strand-break termini. This study represents the first systematic examination of the physical properties of this enzyme. The protein was produced in Escherichia coli as a His-tagged protein, and the purified recombinant protein exhibited both the kinase and the phosphatase activities. The predicted relative molecular mass (M(r)) of the 521 amino acid polypeptide encoded by the sequenced cDNA for PNK and the additional 21 amino acids of the His tag is 59,538. The M(r) determined by low-speed sedimentation equilibrium under nondenaturing conditions was 59,600 +/- 1000, indicating that the protein exists as a monomer, in contrast to T4 phage PNK, which exists as a homotetramer. The size and shape of hPNK in solution were determined by analytical ultracentrifugation studies. The protein was found to have an intrinsic sedimentation coefficient, s(0)(20,w), of 3.54 S and a Stokes radius, R(s), of 37.5 A. These hydrodynamic data, together with the M(r) of 59 600, suggest that hPNK is a moderately asymmetric protein with an axial ratio of 5.51. Analysis of the secondary structure of hPNK on the basis of circular dichroism spectra, which revealed the presence of two negative dichroic bands located at 218 and 209 nm, with ellipticity values of -7200 +/- 300 and -7800 +/- 300 deg x cm(2) x d(mol(-1), respectively, indicated the presence of approximately 50% beta-structure and 25% alpha-helix. Binding of ATP to the protein induced an increase in beta-structure and perturbed tryptophan, tyrosine, and phenylalanine signals observed by aromatic CD and UV difference spectroscopy.
 ...
PMID:Physical properties of human polynucleotide kinase: hydrodynamic and spectroscopic studies. 1166 34

Short-patch repair of DNA single-strand breaks and gaps (SSB) is coordinated by XRCC1, a scaffold protein that recruits the DNA polymerase and DNA ligase required for filling and sealing the damaged strand. XRCC1 can also recruit end-processing enzymes, such as PNK (polynucleotide kinase 3'-phosphatase), Aprataxin and APLF (aprataxin/PNK-like factor), which ensure the availability of a free 3'-hydroxyl on one side of the gap, and a 5'-phosphate group on the other, for the polymerase and ligase reactions respectively. PNK binds to a phosphorylated segment of XRCC1 (between its two C-terminal BRCT domains) via its Forkhead-associated (FHA) domain. We show here, contrary to previous studies, that the FHA domain of PNK binds specifically, and with high affinity to a multiply phosphorylated motif in XRCC1 containing a pSer-pThr dipeptide, and forms a 2:1 PNK:XRCC1 complex. The high-resolution crystal structure of a PNK-FHA-XRCC1 phosphopeptide complex reveals the basis for this unusual bis-phosphopeptide recognition, which is probably a common feature of the known XRCC1-associating end-processing enzymes.
 ...
PMID:Specific recognition of a multiply phosphorylated motif in the DNA repair scaffold XRCC1 by the FHA domain of human PNK. 1915 74

Nitric oxide (NO) causes DNA damage, generating xanthine (Xan, X) and oxanine (Oxa, O) from guanine (Gua, G) and hypoxanthine (Hyp, H) from adenine (Ade, A) by nitrosative oxidation. Although these NO-induced lesions have been thought to cause mutagenic problems in cellular systems, the influence of these lesions on enzymatic functions has not yet been compared systematically. In this study, we investigated the effect of NO-induced lesions on the activities of DNA-binding/recognizing enzymes such as T4 polynucleotide kinase (T4 PNK), DNA ligases (T4 DNA ligase, Taq DNA ligase) and DNA polymerases (E. coli DNA polymerase I, Klenow fragment, T4 DNA polymerase). The phosphorylation efficiencies of T4 PNK are dependent on the base type at the 5'-end of single-stranded DNA, where Oxa congruent with Hyp congruent with Gua > Xan congruent with Ade. The enzymatic reactions efficiencies of DNA ligases or DNA polymerases were observed to be dependent on the base-pairing type bound by the enzymes, where G:C > H:C > O:C > X:C and A:T congruent with H:T > O:T > X:T. These results suggested that NO-induced lesions and their base-pairs could participate in the interaction mechanisms of the DNA-binding/recognizing enzymes in a similar manner as natural nucleobases.
 ...
PMID:Comparison of the molecular influences of NO-induced lesions in DNA strands on the reactivity of polynucleotide kinases, DNA ligases and DNA polymerases. 2009 3

To explore if DNA linkers with 5'-hydroxyl (OH) ends could be joined by commercial T4 and E. coli DNA ligase, these linkers were synthesized by using the solid-phase phosphoramidite method and joined by using commercial T4 and E. coli DNA ligases. The ligation products were detected by using denaturing PAGE silver stain and PCR method. About 0.5-1% of linkers A-B and E-F, and 0.13-0.5% of linkers C-D could be joined by T4 DNA ligases. About 0.25-0.77% of linkers A-B and E-F, and 0.06-0.39% of linkers C-D could be joined by E. coli DNA ligases. A 1-base deletion (-G) and a 5-base deletion (-GGAGC) could be found at the ligation junctions of the linkers. But about 80% of the ligation products purified with a PCR product purification kit did not contain these base deletions, meaning that some linkers had been correctly joined by T4 and E. coli DNA ligases. In addition, about 0.025-0.1% of oligo 11 could be phosphorylated by commercial T4 DNA ligase. The phosphorylation products could be increased when the phosphorylation reaction was extended from 1 hr to 2 hrs. We speculated that perhaps the linkers with 5'-OH ends could be joined by T4 or E. coli DNA ligase in 2 different manners: (i) about 0.025-0.1% of linkers could be phosphorylated by commercial T4 DNA ligase, and then these phosphorylated linkers could be joined to the 3'-OH ends of other linkers; and (ii) the linkers could delete one or more nucleotide(s) at their 5'-ends and thereby generated some 5'-phosphate ends, and then these 5'-phosphate ends could be joined to the 3'-OH ends of other linkers at a low efficiency. Our findings may probably indicate that some DNA nicks with 5'-OH ends can be joined by commercial T4 or E. coli DNA ligase even in the absence of PNK.
 ...
PMID:Detection of ligation products of DNA linkers with 5'-OH ends by denaturing PAGE silver stain. 2276 47

Non-homologous end joining (NHEJ) is critical for the maintenance of genetic integrity and DNA double-strand break (DSB) repair. NHEJ is regulated by a series of interactions between core components of the pathway, including Ku heterodimer, XLF/Cernunnos, and XRCC4/DNA Ligase 4 (Lig4). However, the mechanisms by which these proteins assemble into functional protein-DNA complexes are not fully understood. Here, we show that the von Willebrand (vWA) domain of Ku80 fulfills a critical role in this process by recruiting Aprataxin-and-PNK-Like Factor (APLF) into Ku-DNA complexes. APLF, in turn, functions as a scaffold protein and promotes the recruitment and/or retention of XRCC4-Lig4 and XLF, thereby assembling multi-protein Ku complexes capable of efficient DNA ligation in vitro and in cells. Disruption of the interactions between APLF and either Ku80 or XRCC4-Lig4 disrupts the assembly and activity of Ku complexes, and confers cellular hypersensitivity and reduced rates of chromosomal DSB repair in avian and human cells, respectively. Collectively, these data identify a role for the vWA domain of Ku80 and a molecular mechanism by which DNA ligase proficient complexes are assembled during NHEJ in mammalian cells, and reveal APLF to be a structural component of this critical DSB repair pathway.
 ...
PMID:APLF promotes the assembly and activity of non-homologous end joining protein complexes. 2317 93

In current study, a dual strategy for sensitive detection of T4 polynucleotide kinase (T4 PNK) activity was proposed, which combined split DNAzyme-based background reduction with ligation-triggered DNAzyme cascade for signal amplification. The 8-17 DNAzyme is split into two separate oligonucleotide fragments, which can be separately hybridized to the template DNA to form a ligatable nick after one of the fragments is phosphorylated at the 5at the yl by T4 PNK. With the further addition of Escherichia coli DNA ligase, the two oligonucleotides can be ligated to produce the activated 8-17 DNAzyme, the amount of which is positively related to the activity of T4 PNK. The signal amplification can be achieved through the cyclic cleavage of 8-17 DNAzyme toward the molecular beacon substrate, resulting in an evident fluorescence signal enhancement. The current dual strategy can significantly improve the detection sensitivity of the sensing systems, resulting in a detection limit of 0.001 U mL(-1) for T4 PNK activity, which is superior or comparable to the reported methods. Furthermore, the inhibition effects of adenosine diphosphate and sodium hydrogen phosphate on T4 PNK activity have also been demonstrated with satisfactory results. The current method may be further developed as a universal protocol for monitoring activity and inhibition of nucleotide kinase, and may show the huge potentials in biological process researches, drug discovery, and clinic diagnostics.
 ...
PMID:Highly sensitive detection of T4 polynucleotide kinase activity by coupling split DNAzyme and ligation-triggered DNAzyme cascade amplification. 2438 64