Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:6.5.1.2 (DNA ligase)
2,749 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

DNA with a 5'-adenylpyrophosphoryl cap (5'-adenylated DNA; AppDNA) is an activated form of DNA that is the biochemical intermediate of the reactions catalyzed by DNA ligase, RNA ligase, polynucleotide kinase, and other nucleic acid modifying enzymes. 5'-Adenylated DNA is also useful for in vitro selection experiments. Efficient preparation of 5'-adenylated DNA is therefore desirable for several biochemical applications. Here we have developed a DNA adenylation procedure that uses T4 DNA ligase and is more reliable than a previously reported approach that used the 5'-phosphorylated donor DNA substrate to be adenylated, a DNA template, and ATP but no acceptor strand. Our improved DNA adenylation procedure uses the above components as well as an acceptor strand that has a strategically chosen C-T acceptor-template mismatch directly adjacent to the adenylation site. This mismatch permits adenylation of the donor DNA substrate but largely suppresses subsequent ligation of the donor with the acceptor, as assayed on nine different DNA substrates that collectively have all four DNA nucleotides represented at each of the first two positions. The new DNA adenylation procedure is successful using either laboratory-prepared or commercial T4 DNA ligase and works well on the preparative (2 nmol) scale for all nine of the test DNA substrates.
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PMID:Improvement of DNA adenylation using T4 DNA ligase with a template strand and a strategically mismatched acceptor strand. 1802 69

Nitric oxide (NO) produced in inflamed tissues is known to cause DNA damage by nitrosation or oxidation of base moieties. For investigating the biophysical and biochemical properties of DNA lesions induced by nitric oxide, we prepared synthetic DNA oligomers and analyzed melting temperature (T(m)) or enzymatic recognition of DNA strands containing oxanine (Oxa) and hypoxanthine (Hyp), using guanine (Gua) or adenine (Ade) as control bases, respectively. For enzymatic reaction by DNA-relevant enzymes, we employed T4 polynucleotide kinase, T4 DNA ligase and DNA polymerase (Klenow fragment (exo-)). These analyses revealed that enzymatic recognition of Oxa:Cyt or Hyp:Thy were almost same as Gua:Cyt or Ade:Thy in DNA strands, respectively, indicative of similar molecular and biological recognition of DNA-lesions to normal DNA bases in the cell.
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PMID:Comparison of enzymatic recognition of DNA-duplexes containing NO-induced lesions by DNA-relevant enzymes. 1802 81

Base excision repair (BER) is an evolutionarily conserved process for maintaining genomic integrity by eliminating several dozen damaged (oxidized or alkylated) or inappropriate bases that are generated endogenously or induced by genotoxicants, predominantly, reactive oxygen species (ROS). BER involves 4-5 steps starting with base excision by a DNA glycosylase, followed by a common pathway usually involving an AP-endonuclease (APE) to generate 3' OH terminus at the damage site, followed by repair synthesis with a DNA polymerase and nick sealing by a DNA ligase. This pathway is also responsible for repairing DNA single-strand breaks with blocked termini directly generated by ROS. Nearly all glycosylases, far fewer than their substrate lesions particularly for oxidized bases, have broad and overlapping substrate range, and could serve as back-up enzymes in vivo. In contrast, mammalian cells encode only one APE, APE1, unlike two APEs in lower organisms. In spite of overall similarity, BER with distinct subpathways in the mammals is more complex than in E. coli. The glycosylases form complexes with downstream proteins to carry out efficient repair via distinct subpathways one of which, responsible for repair of strand breaks with 3' phosphate termini generated by the NEIL family glycosylases or by ROS, requires the phosphatase activity of polynucleotide kinase instead of APE1. Different complexes may utilize distinct DNA polymerases and ligases. Mammalian glycosylases have nonconserved extensions at one of the termini, dispensable for enzymatic activity but needed for interaction with other BER and non-BER proteins for complex formation and organelle targeting. The mammalian enzymes are sometimes covalently modified which may affect activity and complex formation. The focus of this review is on the early steps in mammalian BER for oxidized damage.
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PMID:Early steps in the DNA base excision/single-strand interruption repair pathway in mammalian cells. 1816 75

Polymorphisms in a number of genes encoding for DNA repair enzymes have been associated with altering the function of these enzymes and increasing risk of a number of cancers, including colon cancer. We have investigated the association between a common variant in polynucleotide kinase 3' phosphatase (PNKP), a putative DNA repair enzyme, and risk of adenoma recurrence in the Polyp Prevention Trial participants. We also investigated possible interaction or effect modification between carriage of the variant allele, dietary components and risk of adenoma recurrence. Unconditional logistic regression models were used to calculate the odds ratios and 95% confidence intervals for an association between the G/T polymorphism, PNKP T5644G and risk of adenoma recurrence. We observed no association between carriage of the variant allele and risk of adenoma recurrence. Furthermore, we found no effect modification between genotype, dietary components and risk of adenoma recurrence. The PNKP T5644G variant does not seem to be involved in adenoma recurrence in the Polyp Prevention Trial.
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PMID:Polynucleotide kinase 3' phosphatase variant, dietary variables and risk of adenoma recurrence in the Polyp Prevention Trial. 1841 2

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.
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PMID:Specific recognition of a multiply phosphorylated motif in the DNA repair scaffold XRCC1 by the FHA domain of human PNK. 1915 74

In humans, a mutation in the tyrosyl-DNA phosphodiesterase (Tdp1) is responsible for the recessively inherited syndrome spinocerebellar ataxia with axonal neuropathy (SCAN1). Tdp1 is a well-conserved DNA repair enzyme, which processes modified 3' phospho-DNA adducts in vitro. Here, we report that in the yeast Schizosaccharomyces pombe, tdp1 mutant cells progressively accumulate DNA damage and rapidly lose viability in a physiological G0/quiescent state. Remarkably, this effect is independent of topoisomerase I function. Moreover, we provide evidence that Tdp1, with the polynucleotide kinase (Pnk1), processes the same naturally occurring 3'-ends, produced from oxidative DNA damage in G0. We also found that one half of the dead cells lose their nuclear DNA. Nuclear DNA degradation is genetically programmed and mainly depends on the two DNA damage checkpoint responses, ATM/Tel1 and ATR/Rad3, reminiscent to programmed cell death. Diminishing the respiration rate or treating cells with a low concentration of antioxidants rescues the quiescent tdp1 mutant cells. These findings suggest that mitochondrial respiration causes neuronal cell death in the SCAN1 syndrome and in other neurological disorders.
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PMID:Tdp1 protects against oxidative DNA damage in non-dividing fission yeast. 1919 39

Mammalian polynucleotide kinase (mPNK) is a critical DNA repair enzyme whose 5'-kinase and 3'-phoshatase activities function with poorly understood but striking specificity to restore 5'-phosphate/3'-hydroxyl termini at sites of DNA damage. Here we integrated site-directed mutagenesis and small-angle X-ray scattering (SAXS) combined with advanced computational approaches to characterize the conformational variability and DNA-binding properties of mPNK. The flexible attachment of the FHA domain to the catalytic segment, elucidated by SAXS, enables the interactions of mPNK with diverse DNA substrates and protein partners required for effective orchestration of DNA end repair. Point mutations surrounding the kinase active site identified two substrate recognition surfaces positioned to contact distinct regions on either side of the phosphorylated 5'-hydroxyl. DNA substrates bind across the kinase active site cleft to position the double-stranded portion upstream of the 5'-hydroxyl on one side, and the 3'-overhang on the opposite side. The bipartite DNA-binding surface of the mPNK kinase domain explains its preference for recessed 5'-termini, structures that would be encountered in the course of DNA strand break repair.
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PMID:Mechanism of DNA substrate recognition by the mammalian DNA repair enzyme, Polynucleotide Kinase. 1967 25

Oxanine (Oxa), generated from guanine (Gua) by NO- or HNO(2)-induced nitrosative oxidation, has been thought to cause mutagenic problems in cellular systems. In this study, the response of Oxa to different enzymatic functions was explored to understand how similarly it can participate in biomolecular reactions compared to the natural base, Gua. The phosphorylation efficiency of the T4 polynucleotide kinase was highest when Oxa was located on the 5'-end of single stranded DNAs compared to when other nucleobases were in this position. The order of phosphorylation efficiency was as follows; Oxa>Gua>adenine (Ade) approximately thymine (Thy)>cytosine (Cyt). Base-pairing of Oxa and Cyt (Oxa:Cyt) between the ligation fragment and template was found to influence the ligation performance of the T4 DNA ligase to a lesser degree compared to Gua:Cyt. In addition, EcoRI and BglII showed higher cleavage activities on DNA substrates containing Oxa:Cyt than those containing Gua:Cyt, while BamHI, HindIII and EcoRV showed lower cleavage activity; however, this decrease in activity was relatively small.
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PMID:Biomolecular response of oxanine in DNA strands to T4 polynucleotide kinase, T4 DNA ligase, and restriction enzymes. 1990 Apr 15

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.
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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

The cellular response to double-strand breaks (DSBs) in DNA is a complex signalling network, mobilized by the nuclear protein kinase ataxia-telangiectasia mutated (ATM), which phosphorylates many factors in the various branches of this network. A main question is how ATM regulates DSB repair. Here, we identify the DNA repair enzyme polynucleotide kinase/phosphatase (PNKP) as an ATM target. PNKP phosphorylates 5'-OH and dephosphorylates 3'-phosphate DNA ends that are formed at DSB termini caused by DNA-damaging agents, thereby regenerating legitimate ends for further processing. We establish that the ATM phosphorylation targets on human PNKP-Ser 114 and Ser 126-are crucial for cellular survival following DSB induction and for effective DSB repair, being essential for damage-induced enhancement of the activity of PNKP and its proper accumulation at the sites of DNA damage. These findings show a direct functional link between ATM and the DSB-repair machinery.
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PMID:ATM-mediated phosphorylation of polynucleotide kinase/phosphatase is required for effective DNA double-strand break repair. 2163 98


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