Highly efficient DNA synthesis without template and primer DNAs occurs when N.BspD6I DNA nickase is added to a reaction mixture containing deoxynucleoside triphosphates and the large fragment of Bst DNA polymerase. Over a period of 2 h, virtually all the deoxynucleoside triphosphates (dNTPs) become incorporated into DNA. Inactivation of N.BspD6I nickase by heating inhibits DNA synthesis. Optimal N.BspD6I activity is required to achieve high yields of synthesized DNA. Electron microscopy data revealed that the majority of DNA molecules have a branched structure. Cloning and sequencing of the fragments synthesized demonstrated that the DNA product mainly consists of multiple hexanucleotide non-palindromic tandem repeats containing nickase recognition sites. A possible mechanism is discussed that addresses template-independent DNA synthesis stimulated by N.BspD6I nickase.

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A fragment of the 16S RNA of Thermus thermophilus corresponding to the central domain (nucleotides 547-895) has been prepared by transcription in vitro. Incubation of this fragment with the total 30S ribosomal proteins has resulted in the formation of a compact 12S ribonucleoprotein particle. This particle contained five T. thermophilus proteins corresponding to Escherichia coli ribosomal proteins S6, S8, S11, S15, and possibly S18, all of which were previously shown to interact with the central domain of the 16S RNA and to be localized in the platform (side bulge) of the 30S ribosomal subunit. When examined by electron microscopy, isolated particles have an appearance that is similar in size and shape to the corresponding morphological features of the 30S subunit. We conclude that the central domain of the 16S RNA can independently and specifically assemble with a defined subset of ribosomal proteins into a compact ribonucleoprotein particle corresponding to the platform (side bulge) of the 30S subunit.The secondary structure of the 16S RNA of the small 30S ribosomal subunit is divided into three major domains (1). Each domain and a defined set of ribosomal proteins are involved in the formation of different morphological parts of the subunit. The 5Ј domain corresponds to the central body of the 30S subunit, the central domain is involved in the formation of the side bulge or platform, as well as part of the body, and the 3Ј major domain is localized in the head region of the subunit (2-4).It has been shown that fragments of the 16S RNA corresponding to the domains of the 16S RNA are capable of assembling in vitro with specific sets of ribosomal proteins into compact ribonucleoprotein particles (RNPs). Ofengand and coworkers (5) demonstrated formation of the RNP containing the 5Ј domain of the 16S RNA and four ribosomal proteins. Noller and coworkers (6) showed that the fragment of the 16S RNA, corresponding to the 3Ј domain of the 16S RNA, assembles with the group of eight ribosomal proteins. It is noteworthy that the proteins bound in the both complexes are the same as those found associated with these domains in the intact 30S subunit. In this work, we describe the assembly of an RNP containing the central domain of the 16S RNA and five ribosomal proteins of the Thermus thermophilus ribosome.The sequence of the 16S RNA from T. thermophilus was determined, and a high homology of the sequences of the 16S RNA from T. thermophilus and Escherichia coli was shown (7). The homology is 75%, and the computer model of its secondary structure (Fig. 1) does not differ from the model of the E. coli 16S RNA (8). The ribosomal proteins of the 30S subunit of T. thermophilus were purified and identified both by twodimensional gel electrophoresis and amino-terminal sequence analysis (9, 10). The homology of 17 of 19 30S ribosomal proteins from T. thermophilus with corresponding proteins of E. coli was established. Thus the results presented herein are interpretable within the framework of the well-known findings conce...

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