Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

DNA polymerase III', a new form of DNA polymerase III, has been purified 15,000-fold to 90% homogeneity from an Escherichia coli K12 strain. DNA polymerase III's is a subassembly of four subunits of the DNA polymerase III holoenzyme; it has functional and physical properties intermediate between the core DNA polymerase III and holoenzyme. Polyacrylamide gel electrophoresis performed under denaturing conditions indicates DNA polymerase III' to be a complex of the alpha, epsilon, and theta subunits of DNA polymerase III and a newly assigned subunit of the DNA polymerase III holoenzyme, tau (Mr = 83,000). Both gel filtration and phosphocellulose chromatography separate DNA polymerase III from DNA polymerase III'. All enzyme forms can utilize a duplex template containing short gaps. DNA polymerase III', like the DNA polymerase III holoenzyme, can synthesize DNA on a long single-stranded template in the presence of 5 mM spermidine; DNA polymerase III cannot. Alone, DNA polymerase III' is inert in the G4 natural replicative system in which the DNA polymerase III holoenzyme is active. Molecular weight and subunit stoichiometry determinations suggest that DNA polymerase III' contains two units of core DNA polymerase III and two tau subunits.
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PMID:Purification and characterization of DNA polymerase III'. Identification of tau as a subunit of the DNA polymerase III holoenzyme. 703 70

Harnessing CRISPR-Cas9 technology provides an unprecedented ability to modify genomic loci via DNA double-strand break (DSB) induction and repair. We analyzed nonhomologous end-joining (NHEJ) repair induced by Cas9 in budding yeast and found that the orientation of binding of Cas9 and its guide RNA (gRNA) profoundly influences the pattern of insertion/deletions (indels) at the site of cleavage. A common indel created by Cas9 is a 1-bp (+1) insertion that appears to result from Cas9 creating a 1-nt 5' overhang that is filled in by a DNA polymerase and ligated. The origin of +1 insertions was investigated by using two gRNAs with PAM sequences located on opposite DNA strands but designed to cleave the same sequence. These templated +1 insertions are dependent on the X-family DNA polymerase, Pol4. Deleting Pol4 also eliminated +2 and +3 insertions, which are biased toward homonucleotide insertions. Using inverted PAM sequences, we also found significant differences in overall NHEJ efficiency and repair profiles, suggesting that the binding of the Cas9:gRNA complex influences subsequent NHEJ processing. As with events induced by the site-specific HO endonuclease, CRISPR-Cas9-mediated NHEJ repair depends on the Ku heterodimer and DNA ligase 4. Cas9 events are highly dependent on the Mre11-Rad50-Xrs2 complex, independent of Mre11's nuclease activity. Inspection of the outcomes of a large number of Cas9 cleavage events in mammalian cells reveals a similar templated origin of +1 insertions in human cells, but also a significant frequency of similarly templated +2 insertions.
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PMID:CRISPR/Cas9 cleavages in budding yeast reveal templated insertions and strand-specific insertion/deletion profiles. 2978 87


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