Exonuclease endonuclease activity

Schatz O, Mous J, Le Grice SFJ. HIV-1 RT-associated ribonuclease H displays both endonuclease and 3 5 exonuclease activity. EMBO J 1990 9 1171-1176. 

Exonuclease activity In addition to having the 5 —>3 po ) merase activity that synthesizes DNA, and the 3 ->5 exonucleas activity that proofreads the newly synthesized DNA chain lik DNA polymerase III, DNA polymerase I also has a 5 - 3 exon clease activity that is able to hydrolytically remove the RN primer. [Note These activities are exonucleases because the remove one nucleotide at a time from the end of the DNA chaii rather than cleaving it internally as do the endonucleases (Figui 29.18).] First, DNA polymerase I locates the space ("nick between the 3 -end of the DNA newly synthesized by DNA pol] merase III and the 5 -end of the adjacent RNA primer. Next, DN... 

Adenosine 2, 3 -cyclic phosphate is scarcely accumulated, though other nucleoside 2, 3 -cyclic phosphates are accumulated as intermediates. This result suggests that the action of RNase T2 on RNA is owing to the cooperation of an adenylic acid specific endonuclease activity and a nonspecific exonuclease activity releasing mononucleotides from the 3 terminal. 

Nucleases are enzymes that hydrolyze one or more phos-phodiester bonds in nucleic acid polymers. Nucleases may require a free hydroxyl end (exonucleases), with specificity for the 3 or 5 end, or may act only on internal bonds (endonucleases). For example, some probe techniques are based on 5 -exonuclease activity that cleaves nucleic acids between two fluorescent labels. Nucleases can be DNA- or RNA-specific and may act on only double- or single-stranded polymers. For example, DNAse I digests double-stranded DNA (dsDNA) and SI nuclease acts only on smgle-stranded DNA (ssDNA). DNase I can be used to specifically degrade DNA in nucleic acid mixtures when only RNA is of interest. RNAses are very stable enzymes that are common laboratory contaminants. 

Treat the DNA briefly with endonuclease to occasionally nick each strand. Add the polymerase with the radioactive dNTPs. At the broken bond, or nick, the polymerase will degrade the existing strand with its 5 —3 exonuclease activity and replace it with a radioactive complementary copy by using its polymerase activity. This reaction scheme is referred to as nick translation, because the nick is moved, or translated, along the DNA molecule without ever becoming sealed. 

The bacterium Escherichia coli possesses two different AP endonucleases, exonuclease III and endonuclease IV (Ljungquist and Lindahl, 1977). These enzymes define the two known structural classes of AP endonuclease. The predominant enzyme in E. coli is exonuclease III, which has 3 5 exonuclease activity in addition to the characteristic AP endonuclease activities detailed above (Rogers and Weiss, 1980 Weiss, 1976). Endonuclease IV, whose expression in E. coli is induced in response to oxidative stress (Chan and Weiss, 1987), is homologous to the m or AP endonuclease found in baker s yeast, APNl (Popoff et al, 1990). The m or human AP endonuclease, APEl (also known as Ref-1 or HAPl), is homologous to exonuclease III (Demple et al, 1991). APEl is multifunctional, possessing redox-dependent transcriptional activation (Xanthoudakis et al, 1992) and acetylation-dependent, redox-independent transcriptional repression activities (Bhakat et al, 2003 Okazaki et al, 1994) in addition to AP... 

One assay we use to monitor exonuclease activity was adapted from a published method that employs uniformly labeled DNA. Using double-stranded labeled DNA templates, we can determine specificity by measuring whether cpms increase (5 - -3 exonuclease) or decrease (3 ->5 exonuclease) upon the addition of dNTPs (10-100 pJl ). For specifically monitoring DNA polymerases with 5 3 structure-specific endonuclease activity, duplex DNA templates which contain displaced 5 ends are preferred. As is the case for polymerase activity measurements, exonuclease assays are significantly influenced by reaction conditions, and salt concentration, incubation temperature, and DNA concentration should be specifically optimized for each DNA polymerase. 

Exonuclease III (Exo III) of E. coli is a monomeric multifunctional enzyme (31 kDa) that catalyzes the hydrolysis of at least four different types of phosphoester bonds in dsDNA (Fig. 3.4). The main enzymatic activity of Exo III is the 3 — 5 -exonuclease activity that carries out the successive release of 5 -P-mononucleotides from the 3 ends of dsDNA. The second activity is the DNA 3 -phosphatase activity that hydrolyzes 3 -terminal phosphomonoesters. In fact, Exo III was initially discovered as a DNA 3 -phosphatase in E. coli (1,2). Exo III has a third activity which degrades the RNA strand in a DNA RNA heteroduplex, thus the RNase H activity. The fourth activity of Exo III is an AP endonuclease which cleaves phosphodiester bonds at apurinic or apyrimidinic sites. 

The structural gene for Exo III is xthA (7,36), which is located at the 38-min position on the E. coli map. A number of xth mutants ts and deletion) have been isolated, all of which simultaneously affect the AP endonuclease, 3 -phosphatase, and exonuclease activities. 

Wild-type E. coli K12 is estimated to contain —3500 Exo 111 molecules, which are responsible for —85% of the total cellular exonuclease activity, over 90% of the total 3 -phosphomonoesterase activity, and more than 80% of the total cellular AP endonuclease activity. 

Retroviral RTases are multifunctional enzymes that exhibit at least three distinct enzymatic activities (i) RNA-directed DNA polymerase, (ii) DNA-directed DNA polymerase in the conversion of ssDNA to dsDNA, and (iii) RNase H that selectively removes the RNA moiety from a RNA DNA heteroduplex. Reverse transcriptases lack both 3 5 - and 5 —> 3 -exonuclease activities, and exhibit relatively high frequencies of nucleotide misincorporation. The RNA- and DNA-dependent polymerase activities are physically inseparable. Reverse transcriptases from avian retroviruses, but not from mammalian retroviruses, exhibit a fourth activity, DNA endonuclease, that is essential for the integration of the dsDNA intermediate into the host chromosome and for the productive infection of the retrovirus. 

The reverse transcriptase from the avian myeloblastosis virus (AMV RTase) is a prototype of avian retroviral RTases. The enzyme is a dimer (155 kDa) composed of nonidentical subunits a and /3. AMV RTase has multiple enzymatic activities including RNA-directed DNA polymerase (reverse transcriptase), DNA-directed DNA polymerase, RNase H, and DNA endonuclease. AMV RTase does not have 3 5 - or 5 3 -exonuclease activity. AMV RTase is a key reagent... 

Utilising a reversion assay in Salmonella enterica, Prieto et al reported an increased frequency of point mutations following bile-salt exposure. Mutations were predominantly nucleotide substitutions (GC to AT transitions) and -1 frameshift mutations.The frameshifts were dependent on SOS induction and linked to the activity of DinB polymerase (Pol IV). The authors proposed that the GC to AT transitions stimulated by bile, could have arisen from oxidative processes giving rise to oxidised cytosine residues. Consistent with this hypothesis, the authors demonstrated that strains of S. enterica-lacking enzymes required for base-excision repair (endonuclease III and exonuclease IV) and the removal of oxidised bases, demonstrated increased bile-acid sensitivity compared with competent strains. In another study using E. coli, resistance to the DNA-damaging effects of bile was associated with Dam-directed mismatch repair, a pathway also involved with the repair of oxidative DNA lesions. ... 

Lacks and Greenberg have partially purified an endonuclease from Dip-lococcus pneumoniae in conjunction with the exonuclease cited in Section II,A (10). This enzyme is active on both native and denatured DNA and produces 5 -phosphoryl-terminated olingonucleotides. 

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