Site-Specific Photomodification of Single-Stranded DNA Targets by Arylazide and Perfluoroarylazide Derivatives of Oligonucleotides

ABSTRACT The Rep68 and Rep78 proteins (Rep68/78) of adeno-associated virus type 2 (AAV) are critical for AAV replication and site-specific integration. They bind specifically to the AAV inverted terminal repeats (ITRs) and possess ATPase, helicase, and strand-specific/site-specific endonuclease activities. In the present study, we further characterized the AAV Rep68/78 helicase, ATPase, and endonuclease activities by using a maltose binding protein-Rep68 fusion (MBP-Rep68Δ) produced in Escherichia coli cells and Rep78 produced in vitro in a rabbit reticulocyte lysate system. We found that the minimal length of single-stranded DNA capable of stimulating the ATPase activity of MBP-Rep68Δ is 100 to 200 bases. The degree of stimulation correlated positively with the length of single-stranded DNA added to the reaction mixture. We then determined the ATP concentration needed for optimal MBP-Rep68Δ helicase activity and showed that the helicase is active over a wide range of ATP concentrations. We determined the directionality of MBP-Rep68Δ helicase activity and found that it appears to move in a 3′ to 5′ direction, which is consistent with a model in which AAV Rep68/78 participates in AAV DNA replication by unwinding DNA ahead of a cellular DNA polymerase. In this report, we also demonstrate that single-stranded DNA is capable of inhibiting the MBP-Rep68Δ or Rep78 endonuclease activity greater than 10-fold. In addition, we show that removal of the secondary Rep68/78 binding site, which is found only in the hairpin form of the AAV ITR, causes a three- to eightfold reduction in the ability of the ITR to be used as a substrate for the Rep78 or MBP-Rep68Δ endonuclease activity. This suggests that contact between Rep68/78 and this secondary element may play an important role in the Rep-mediated endonuclease activity.

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Replicase, Excisionase, and Integrase Genes of theStreptomyces Element pSAM2 Constitute an Operon Positively Regulated by the pra Gene

Journal of Bacteriology ◽

10.1128/jb.180.12.3056-3061.1998 ◽

1998 ◽

Vol 180 (12) ◽

pp. 3056-3061 ◽

Cited By ~ 23

Author(s):

Guennadi Sezonov ◽

Anne-Marie Duchêne ◽

Annick Friedmann ◽

Michel Guérineau ◽

Jean-Luc Pernodet

Keyword(s):

Streptomyces Lividans ◽

Sequence Analyses ◽

Site Specific ◽

Streptomyces Ambofaciens ◽

A Site ◽

Derivatives Of

ABSTRACT pSAM2 is a site-specific integrative element fromStreptomyces ambofaciens. The pra gene described earlier as an activator of pSAM2 replication is shown here to be also involved in the activation of its integration and excision. This was evidenced with derivatives of pSAM2 mutant B3 in which thepra gene was placed under the control of the inducibletipAp promoter. Transformation of Streptomyces lividans by these derivatives was efficient only whenpra expression was induced, indicating its involvement in pSAM2 integration activation. Once established, these constructions remained integrated in the chromosome under noninduced conditions. Activation of the pra expression provoked strong activation of their excision, leading to the appearance of free forms. The results of functional, transcriptional, and sequence analyses allowed to conclude that the three genes repSA, xis, andint coding for the pSAM2 replicase, excisionase, and integrase, respectively, constitute an operon directly or indirectly activated by pra.

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Eleven tungsten atom cluster labels: high-resolution, site-specific probes for electron microscopy.

Journal of Histochemistry & Cytochemistry ◽

10.1177/38.12.1701458 ◽

1990 ◽

Vol 38 (12) ◽

pp. 1787-1793 ◽

Cited By ~ 10

Author(s):

J F Hainfeld ◽

C J Foley ◽

L E Maelia ◽

J J Lipka

Keyword(s):

Electron Microscopy ◽

Electron Microscope ◽

High Resolution ◽

Sulfonyl Chloride ◽

Sulfhydryl Groups ◽

Tungsten Atom ◽

Site Specific ◽

Atom Cluster ◽

Derivatives Of

Two derivatives of a tungstate cluster containing 11 tungsten atoms (W11PO39SiR4-) have been synthesized which enable them to be covalently attached to biomolecules at specific sites. The tungstate cluster is 1.0 nm in diameter, electron dense, and visible in the electron microscope. One derivative is a W11-sulfonyl chloride, reactive with amines and sulfhydryls. The second compound is a W11-thiosulfonate which can be used to label sulfhydryl groups. These new labels are beam resistant and provide significantly higher resolution then most other electron microscopy (EM) markers. Labeling of the protein albumin is described as an example.

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