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)

A new procedure has been developed for the efficient cloning of complex PCR mixtures, resulting in libraries exclusively consisting of recombinant clones. Recombinants are generated between PCR products and a PCR-amplified plasmid vector. The procedure does not require the use of restriction enzymes, T4 DNA ligase or alkaline phosphatase. The 5'-ends of the primers used to generate the cloneable PCR fragments contain an additional 12 nucleotide (nt) sequence lacking dCMP. As a result, the amplification products include 12-nt sequences lacking dGMP at their 3'-ends. The 3'-terminal sequence can be removed by the action of the (3'----5') exonuclease activity of T4 DNA polymerase in the presence of dGTP, leading to fragments with 5'-extending single-stranded (ss) tails of a defined sequence and length. Similarly, the entire plasmid vector is amplified with primers homologous to sequences in the multiple cloning site. The vector oligos have additional 12-nt tails complementary to the tails used for fragment amplification, permitting the creation of ss-ends with T4 DNA polymerase in the presence of dCTP. Circularization can occur between vector molecules and PCR fragments as mediated by the 12-nt cohesive ends, but not in mixtures lacking insert fragments. The resulting circular recombinant molecules do not require in vitro ligation for efficient bacterial transformation. We have applied the procedure for the cloning of inter-ALU fragments from hybrid cell-lines and human cosmid clones.
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PMID:Ligation-independent cloning of PCR products (LIC-PCR). 223 90

varphiX174 RF (replicative form) II DNA, labeled in vivo with [methyl-(3)H]thymidine, was isolated from Escherichia coli polA (DNA polymerase I-deficient) and polA(+) cells during RF replication. [(32)P]dCMP was incorporated into the gaps present in the RF II DNA with [alpha-(32)P]dCTP and T4 DNA polymerase. Sedimentation in alkaline sucrose gradients revealed that much of the incorporated (32)P was present in a heterogeneous collection of fragments shorter than unit length. Inclusion of polynucleotide ligase in the gap-filling reaction increased the average size of the (32)P-labeled fragments. Gel electrophoresis of the products formed by digestion of the (32)P-labeled RF II molecules with the restriction nuclease, endonuclease R, indicated that in the population of RF II molecules gaps could occur anywhere in the genome. Competition-annealing experiments provided evidence that the majority of the label incorporated into gaps was present in the minus strand. RF II molecules isolated from polA(+) cells were enriched for gaps in a unique region of the genome in comparison with RF II molecules isolated from polA cells. The presence of multiple gaps in the minus strand implies that it is synthesized by a discontinuous mechanism during varphiX RF replication.
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PMID:Structure of nascent phiX174 replicative form: evidence for discontinuous DNA replication. 452 6

Linear phiX174 single-stranded DNA can be isolated from phiX phage particles produced under various conditions. About half of the linear strands have a dGMP residue at the 5' end, the remaining have roughly comparable amounts of dCMP, dTMP, and dAMP. The linear strands can be converted to covalently closed circular molecules by polynucleotide ligase, but only after they have been incubated with T4 DNA polymerase and deoxynucleoside triphosphates. Experiments with endonuclease R, the restriction enzyme from Haemophilus influenzae, indicated that the nucleotides incorporated into the DNA during this reaction were found predominantly in a limited region of the genome. The results suggest that the normal intermediate in single-stranded phiX174 DNA synthesis may be a single-stranded linear molecule which is shorter than unit length and is intrinsically capable of circularization.
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PMID:Mechanism of replication of single-stranded PhiX174 DNA. VII. Circularization of the progeny viral strand. 459 Oct 49

The G:U mismatch in genomic DNA mainly arises from deamination of cytosine residues and is repaired by the base excision repair pathway. We found that a bovine testis crude nuclear extract conducts uracil-initiated base excision repair in vitro. A 51-base pair synthetic DNA substrate containing a single G:U mismatch was used, and incorporation of dCMP during repair was exclusively to replace uracil. A neutralizing polyclonal antibody against DNA polymerase beta (beta-pol) inhibited the repair reaction. ddCTP also inhibited the repair reaction, whereas aphidicolin had no significant effect, suggesting that activity of beta-pol was required. Next, the base excision repair system was reconstituted using partially purified components. Several of the enzymatic activities required were resolved, such that DNA ligase and the uracil-DNA glycosylase/apurinic/apyrimidinic endonuclease activities were separated from the DNA polymerase requirement. We found that purified beta-pol could restore full DNA repair activity to the DNA polymerase-depleted fraction, whereas purified DNA polymerases alpha, delta, and epsilon could not. These results with purified proteins corroborated results obtained with the crude extract and indicate that beta-pol is responsible for the single-nucleotide gap filling reaction involved in this in vitro base excision repair system.
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PMID:DNA polymerase beta conducts the gap-filling step in uracil-initiated base excision repair in a bovine testis nuclear extract. 782 35

Here we present CapSelect as a novel experimental approach for the selective enrichment of full-length cDNAs in PCR-mediated analysis of mRNA sequences. The method combines the 5'-CAP-dependent addition of specifically three to four non-templated dCMP residues to the 3'-end of full-length cDNAs by reverse transcriptases in the presence of manganese and the controlled ribonucleotide tailing of cDNA ends by terminal deoxynucleotidyl transferase using rATP. By virtue of the generated terminal sequence motif (5'-dC(3-4)rA(3-4)), full-length cDNAs are selectively anchored to a double-stranded DNA adapter (with a dT(3-4)dG(3)3'-overhang) by T4 DNA ligase. The technique described is highly efficient, discriminates premature termination products and enriches full-length cDNAs.
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PMID:CapSelect: a highly sensitive method for 5' CAP-dependent enrichment of full-length cDNA in PCR-mediated analysis of mRNAs. 1051 26

Pol lambda is a DNA repair enzyme with a high affinity for dNTPs, an intrinsic dRP lyase activity, a BRCT domain involved in interactions with NHEJ factors, and also capable to interact with the PCNA processivity factor. Based on this potential, Pol lambda could play a role in BER, V(D)J recombination, NHEJ and TLS. Here we show that human Pol lambda uses a templating 7,8-dihydro-8-oxoguanine (8oxoG) base, a common mutagenic form of oxidative damage, as efficiently as an undamaged dG, but giving rise to the alternative insertion of either dAMP or dCMP. However, Pol lambda strongly discriminated against the extension of the mutagenic 8oxoG:dAMP pair. Conversely, Pol lambda readily extended the non-mutagenic 8oxoG:dCMP pair with an efficiency that was even higher than that displayed on undamaged dG:dCMP pair. A similar capacity for non-mutagenic extension was also shown to occur in the case of O6-methylguanine (m6G), a mutagenic and cytotoxic DNA adduct. A comparison of these novel properties of human Pol lambda with those of other DNA polymerases involved in TLS will be discussed. Interestingly, when double-strand breaks are associated to base damage, modifications as 8oxoG could be eventually part of the synapsis required to join ends, and therefore, the capacity of Pol lambda either to insert opposite 8oxoG or to extend correct base pairs containing such a damage could be beneficial for its role in NHEJ.
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PMID:Human DNA polymerase lambda is a proficient extender of primer ends paired to 7,8-dihydro-8-oxoguanine. 1768 65