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:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is an important tobacco-specific carcinogen associated with lung cancer. Its complex enzymatic activation, leading to methyl and pyridyloxobutyl (POB)-modified DNA, makes DNA damage difficult to characterize and quantify. Therefore, we use the NNK analogue 4-[(acetoxymethyl)nitrosamino]-1-(3-pyridyl)-1-butanone (NNKOAc) to induce damage in genomic DNA, and to map the sites and frequency of adducts at nucleotide resolution using ligation-mediated polymerase chain reaction and terminal transferase-dependent polymerase chain reactions (LMPCR and TDPCR). NNKOAc induced single-strand breaks in a concentration-dependent manner. Post-alkylation treatments, including hot piperidine or digestion with the enzymes Escherichia coli 3-methyladenine-DNA glycosylase II, formamidopyrimidine-DNA glycosylase, Escherichia coli endonuclease III, or phage T4 UV endonuclease V did not increase the level of DNA breaks in NNKOAc-treated DNA. Detection of DNA damage using LMPCR was possible only when POB-DNA was 5'-phosphorylated prior to the LMPCR procedure. NNKOAc generated damage at all four bases with the decreasing order guanine>adenine>cytosine>thymine. In contrast to NNKOAc damage distribution patterns, those induced by N-nitroso(acetoxymethyl)methylamine, a methylating NNK analog, induced damage principally at G positions detectable by enzymatic means that did not require phosphorylation. Analysis of damage distribution patterns, reveals a high frequency of damage in the p53 gene in codons 241 and 245 and a lower frequency of damage in codon 248. We analyzed the 3' termini of the NNKOAc induced single-strand breaks using a (32)P-post-labeling assay or a nucleotide exchange reaction at the 3'-termini catalyzed by T4 DNA polymerase combined with endonuclease IV treatment. Both methods indicate that the 3' termini of the single-strand breaks are not hydroxyl groups and are blocked by an unknown chemical structure that is not recognized by endonuclease IV. These data are consistent with POB-phosphotriester hydrolysis leading to strand breaks in DNA. The POB-damage could be mutagenic because NNKOAc produces single-strand breaks with the products being a 5'-hydroxyl group and a 3'-blocking group and strand breaks. These results represent the first step in determining if NNK pyridyloxobutylates DNA with sequence specificity similar to those observed with other model compounds.
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PMID:Characterization and mapping of DNA damage induced by reactive metabolites of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) at nucleotide resolution in human genomic DNA. 1167 38

The crystal structure of the catalytic core of murine terminal deoxynucleotidyltransferase (TdT) at 2.35 A resolution reveals a typical DNA polymerase beta-like fold locked in a closed form. In addition, the structures of two different binary complexes, one with an oligonucleotide primer and the other with an incoming ddATP-Co(2+) complex, show that the substrates and the two divalent ions in the catalytic site are positioned in TdT in a manner similar to that described for the human DNA polymerase beta ternary complex, suggesting a common two metal ions mechanism of nucleotidyl transfer in these two proteins. The inability of TdT to accommodate a template strand can be explained by steric hindrance at the catalytic site caused by a long lariat-like loop, which is absent in DNA polymerase beta. However, displacement of this discriminating loop would be sufficient to unmask a number of evolutionarily conserved residues, which could then interact with a template DNA strand. The present structure can be used to model the recently discovered human polymerase mu, with which it shares 43% sequence identity.
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PMID:Crystal structures of a template-independent DNA polymerase: murine terminal deoxynucleotidyltransferase. 1182 35

A new gene (POLL), has been identified encoding the novel DNA polymerase lambda and mapped to mouse chromosome 19 and at human chromosome 10. DNA polymerase lambda contains all the critical residues involved in DNA binding, nucleotide binding, nucleotide selection, and catalysis of DNA polymerization and has been assigned to family X based on sequence homology with polymerase beta, lambda, mu, and terminal deoxynucleotidyltransferase. Here we describe a purification of DNA polymerase lambda from calf thymus that preferentially can replicate damaged DNA. By testing polymerase activity on non-damaged and damaged DNA, DNA polymerase lambda was purified trough five chromatographic steps to near homogeneity and identified as a 67-kDa polypeptide that cross-reacted with monoclonal antibodies against DNA polymerase beta and polyclonal antibodies against DNA polymerase lambda. DNA polymerase lambda had no detectable nuclease activities and, in contrast to DNA polymerase beta, was aphidicolin-sensitive. DNA polymerase lambda was a 6-fold more accurate enzyme in an M13mp2 forward mutation assay and 5-fold more accurate in an M13mp2T90 reversion system than human recombinant DNA polymerase beta. The biochemical properties of the calf thymus DNA polymerase lambda, described here for the first time, are discussed in relationship to the proposed role for this DNA polymerase in vivo.
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PMID:DNA polymerase lambda from calf thymus preferentially replicates damaged DNA. 1188 60

Terminal deoxynucleotidyl transferase (TdT), a template-independent DNA polymerase, contributes to antigen receptor diversity in lymphocytes. Using in situ hybridization, we found that tdt is expressed within neurons of the adult mouse brain. tdt mRNA was localized within pyramidal neurons in the hippocampus, granule and polymorphic cells in the dentate gyrus, Purkinje neurons in the cerebellum, and cortical cells. Increased levels of tdt mRNA in the hippocampus, neocortex, and cerebellum were associated with rearing C57BL/6 mice, but not DBA/2 mice, in enriched environments. Unlike wild types (WT), tdt (-/-) mice did not show improvement in spatial learning and memory as a result of rearing in enriched environments. These results suggest that tdt may be involved in learning and memory saving.
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PMID:Experience-dependent expression of terminal deoxynucleotidyl transferase in mouse brain. 1282 97

DNA polymerase mu (Pol mu) is a novel family X DNA polymerase that has been suggested to play a role in micro-homology mediated joining and repair of double strand breaks. We show here that human Pol mu is not able to discriminate against the 2'-OH group of the sugar moiety. It inserts rNTPs with an efficiency that is <10-fold lower than that of dNTPs, in sharp contrast with the >1000-fold discrimination characteristic of most DNA-dependent DNA polymerases. The lack of sugar discrimination by Pol mu is demonstrated by its ability to add rNTPs to both DNA and RNA primer strands, and to insert both deoxy- and ribonucleotides on growing nucleic acid chains. 3D-modelling of human Pol mu based on the available Pol beta and TdT structural information allowed us to predict candidate residues involved in sugar discrimination. Thus, a single amino acid substitution in which Gly433 residue of Pol mu was mutated to the consensus tyrosine present in Pol beta, produced a strong increase in the discrimination against ribonucleotides. The unusual capacity to insert both rNTPs and dNTPs will be discussed in the context of the predicted roles of Pol mu in DNA repair.
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PMID:Lack of sugar discrimination by human Pol mu requires a single glycine residue. 1288 4

Upon isolation of DNA from normal eukaryotic cells by standard methods involving extensive proteolytic treatment, a rather homogeneous population of loop-size, double-stranded DNA fragments is regularly obtained. These DNA molecules can be efficiently end-labeled by the DNA polymerase I Klenow fragment, as well as by a 3'- to -5'-exonuclease-free Klenow enzyme, but not by terminal transferase (TdT) unless the ends have been filled up by Klenow, suggesting that dominantly 5' protruding termini are generated upon fragmentation. The filled-up termini were used for cloning the distal parts of the approximately 50 kb fragments. BLAST analysis of the sequence of several clones allowed us to determine the sequence of the non-cloned side of the breakpoints. Comparison of 25, 600 bp-long breakpoint sequences demonstrated prevalence of repetitive elements. Consensus motives characteristic of the breakpoint sequences have been identified. Several sequences exhibit peculiar computed conformational characteristics, with sharp transition or center of symmetry located exactly at the breakpoint. Our data collectively suggest that chromatin fragmentation involves nucleolytic cleavages at fragile/hypersensitive sites delimiting loop-size fragments in a non-random manner. Interestingly, the sequence characteristics of the breakpoints are reminiscent of certain breakpoint cluster regions frequently subject to gene rearrangements.
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PMID:Non-random features of loop-size chromatin fragmentation. 1289 17

DNA polymerase (pol) lambda is homologous to pol beta and has intrinsic polymerase and terminal transferase activities. However, nothing is known about the amino acid residues involved in these activities. In order to precisely define the nucleotide-binding site of human pol lambda, we have mutagenised two amino acids, Tyr505 and the neighbouring Phe506, which were predicted by structural homology modelling to correspond to the Tyr271 and Phe272 residues of pol beta, which are involved in nucleotide binding. Our analysis demonstrated that pol lambda Phe506Arg/Gly mutants possess very low polymerase and terminal transferase activities as well as greatly reduced abilities for processive DNA synthesis and for carrying on translesion synthesis past an abasic site. The Tyr505Ala mutant, on the other hand, showed an altered nucleotide binding selectivity to perform the terminal transferase activity. Our results suggest the existence of a common nucleotide-binding site for the polymerase and terminal transferase activities of pol lambda, as well as distinct roles of the amino acids Tyr505 and Phe506 in these two catalytic functions.
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PMID:Mutagenesis of human DNA polymerase lambda: essential roles of Tyr505 and Phe506 for both DNA polymerase and terminal transferase activities. 1462 24

The addition of nontemplated (N) nucleotides to coding ends in V(D)J recombination is the result of the action of a unique DNA polymerase, TdT. Although N-nucleotide addition by TdT plays a critical role in the generation of a diverse repertoire of Ag receptor genes, the mechanism by which TdT acts remains unclear. We conducted a structure-function analysis of the murine TdT protein to determine the roles of individual structural motifs that have been implicated in protein-protein and protein-DNA interactions important for TdT function in vivo. This analysis demonstrates that the N-terminal portion of TdT, including the BRCA-1 C-terminal (BRCT) domain, is not required for TdT activity, although the BRCT domain clearly contributes quantitatively to N-nucleotide addition activity. The second helix-hairpin-helix domain of TdT, but not the first, is required for activity. Deletional analysis also suggested that the entire C-terminal region of TdT is necessary for N-nucleotide addition in vivo. The long isoform of TdT was found to reduce N-nucleotide addition by the short form of TdT, but did not increase nucleotide deletion from coding ends in either human or rodent nonlymphoid cells. We consider these results in light of the recently reported structure of the catalytic region of TdT.
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PMID:Mutational analysis of terminal deoxynucleotidyltransferase-mediated N-nucleotide addition in V(D)J recombination. 1510 Feb 89

DNA polymerase lambda contains template-dependent (DNA polymerase) and template-independent (terminal transferase) activities. In this study we enzymologically characterized the terminal transferase activity of polymerase lambda (pol lambda-tdt). Pol lambda-tdt activity was strongly influenced by the nature of the 3'-terminal sequence of the DNA substrate, and it required a single-stranded (ss) DNA 3'-overhang of about 9-12 nucleotides for optimal activity. The strong preference observed for pyrimidine versus purine nucleotide incorporation was found to be due, at least partially, to a steric block imposed by the residue Tyr-505 in the active site of pol lambda. Pol lambda-tdt was found to be able to elongate a 3'-ssDNA end by two alternative mechanisms: first, a template-independent one resulting in addition of 1 or 2 nucleotides, and second, a template-dependent one where a homopolymeric tract as short as 3 nucleotides at the 3'-end could be used as a template to direct DNA polymerization by a looping back mechanism. Furthermore repetitive cycles of DNA synthesis resulted in the expansion of such a short homopolymeric terminal sequence. Most importantly we found that the proliferating cell nuclear antigen was able to selectively block the looping back mechanism while stimulating the single terminal nucleotide addition. Finally replication protein A completely suppressed the transferase activity of pol lambda while stimulating the polymerase activity, suggesting that proliferating cell nuclear antigen and replication protein A can coordinate the polymerase and the terminal transferase activities of pol lambda.
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PMID:DNA elongation by the human DNA polymerase lambda polymerase and terminal transferase activities are differentially coordinated by proliferating cell nuclear antigen and replication protein A. 1553 31

Nonhomologous end joining (NHEJ) is a major pathway in multicellular eukaryotes for repairing double-strand DNA breaks (DSBs). Here, the NHEJ reactions have been reconstituted in vitro by using purified Ku, DNA-PK(cs), Artemis, and XRCC4:DNA ligase IV proteins to join incompatible ends to yield diverse junctions. Purified DNA polymerase (pol) X family members (pol mu, pol lambda, and TdT, but not pol beta) contribute to junctional additions in ways that are consistent with corresponding data from genetic knockout mice. The pol lambda and pol mu contributions require their BRCT domains and are both physically and functionally dependent on Ku. This indicates a specific biochemical function for Ku in NHEJ at incompatible DNA ends. The XRCC4:DNA ligase IV complex is able to ligate one strand that has only minimal base pairing with the antiparallel strand. This important aspect of the ligation leads to an iterative strand-processing model for the steps of NHEJ.
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PMID:A biochemically defined system for mammalian nonhomologous DNA end joining. 1557 26


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