Klenow fragment of Escherichia coli DNA polymerase I, which was cocrystallized with duplex DNA, positioned 11 base pairs of DNA in a groove that lies at right angles to the cleft that contains the polymerase active site and is adjacent to the 3' to 5' exonuclease domain. When the fragment bound DNA, a region previously referred to as the "disordered domain" became more ordered and moved along with two helices toward the 3' to 5' exonuclease domain to form the binding groove. A single-stranded, 3' extension of three nucleotides bound to the 3' to 5' exonuclease active site. Although this cocrystal structure appears to be an editing complex, it suggests that the primer strand approaches the catalytic site of the polymerase from the direction of the 3' to 5' exonuclease domain and that the duplex DNA product may bend to enter the cleft that contains the polymerase catalytic site.

http://science.sciencemag.org/content/sci/260/5106/352.full.pdf

Structure of DNA polymerase I Klenow fragment bound to duplex DNA

G e n e t i c molecular markers are DNA segments that behave as landmarks for genome analysis. These segments usually represent variant or polymorphic sites that can be identified using general strategies such as molecular hybridization or enzymatic amplification of DNA. For years, DNA-based diagnostic markers have been used in general organismal identification and in the construction of genetic linkage maps. (1) The widely used restriction fragment length polymorphisms (RFLPs), for example, are molecular markers identified by endonuclease restriction and blot hybridization of DNA. (z) DNA amplification using PCR (3'4) has also been used extensively in many applications to study polymorphic loci, like hypervariable minisatellites (s) or microsatellites harboring simple sequence repeats, (6-8) and to generate sequence-tagged sites (STSs) (9) for genetic and physical mapping. Several strategies involving DNA replication, DNA ligation, or RNA transcription have been used for in vitro amplification of nucleic acids. (1~ However, PCR remains the most widely accepted amplification tool. Primer-directed amplification of DNA, at first used in PCR to amplify cognate regions present at very low levels in the genome, has extended DNA analysis to regions adjacent to sequenced DNA segments, to unknown DNA, and even to the study of RNA-expressed sequences. (~s-~7) Amplification strategies can be grouped according to the mechanism of the amplification process (Table 1). Amplification with specific primers in the PCR, for example, is a determinate process that requires prior knowledge of the template sequence and targets usually one defined amplification site. Similarly, amplification of interspersed repetitive sequences (IRS), like Alu-PCR (18) or REP-PCR, (19) are determinate processes that target multiple sites of defined sequence in both DNA strands. In contrast, amplification with degenerate primers in random indeterminate amplification processes, like random primed amplification (RPA), (2~ primer-extension preamplification (PEP), (21) and random PCR (rPCR), (22) take advantage of stochastic annealing events that randomly amplify nucleic acid stretches or even whole genomes by targeting sites of a noncognate nature. These random DNA amplification procedures are generally used for radioactive or fluorescent labeling of nucleic acids, to increase the amount of DNA in the construction of representative cDNA libraries, or for PCR typing of single haploid cells. Other kinds of amplification processes, although still determinate, do not require prior knowledge of template sequence and can target single or multiple sites in a genome or template molecule. These strategies can use either one arbitrary primer in conjunction with a specific primer (23) or one or more arbitrary primers (24-26) to study single or multiple amplicons, respectively, and can even be extended to the analysis of RNA populations. (27-28) In particular, strategies like gene-walking PCR (23) and differential cDNA PCR (27) study specific template regions arbitrary in length but juxtaposed to a known DNA segment defining a specific primer. Like anchored PCR, (29) these hemispecific reactions allow analysis of unknown genomic regions corresponding to mRNA sequences, or adjacent to STSs or sequenced stretches of DNA.

/pdf/amplifying-dna-with-arbitrary-oligonucleotide-primers-mdkxecylp4.pdf

Amplifying DNA with arbitrary oligonucleotide primers.

The ribosome accelerates the rate of peptide bond formation by at least 10(7)-fold, but the catalytic mechanism remains controversial. Here we report evidence that a functional group on one of the tRNA substrates plays an essential catalytic role in the reaction. Substitution of the P-site tRNA A76 2' OH with 2' H or 2' F results in at least a 10(6)-fold reduction in the rate of peptide bond formation, but does not affect binding of the modified substrates. Such substrate-assisted catalysis is relatively uncommon among modern protein enzymes, but it is a property predicted to be essential for the evolution of enzymatic function. These results suggest that substrate assistance has been retained as a catalytic strategy during the evolution of the prebiotic peptidyl transferase center into the modern ribosome.

Substrate-assisted catalysis of peptide bond formation by the ribosome

The nucleotide sequence of the rpoB gene of Salmonella typhimurium has been determined in this work. It was compared with known sequences of the gene from other sources and the conservative regions were detected. This allowed some interesting conclusions to be made about the distribution of the functional domains in bacterial RNA polymerase and about the three-dimensional structure of its β subunit.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1988.tb14384.x

Genes coding for RNA polymerase β subunit in bacteria

Structural Organization of the RNA Polymerase-Promoter Open Complex

A method is proposed for localization of the sites of affinity labelling of the β subunit of Escherichia coli RNA polymerase. The principle of this method is similar to that of the methods of rapid sequencing of nucleic acids.



The polypeptide bearing a radioactive affinity label at one of the amino acid residues is subjected to short-term treatment with cyanogen bromide. The conditions of this reaction are selected in such a way that less than one cleavage occurs on average per polypeptide chain. Two series of radioactive peptides are formed, one involving all the possible N-terminal peptides and the other the C-terminal peptides. The distribution of the lengths of these peptides is studied by means of gel electrophoresis and compared with the theoretical ones based on the known amino acid sequence of the β subunit. Obviously, the affinity label resides between the C-terminus of the shortest N-terminal radioactive peptide and the N-terminus of the shortest C-terminal radioactive peptide. In order to increase reliability and resolution of the method, partial trypsinolysis may be employed.



The evidence obtained suggests that lysine residues over the regions 1036–1066, 1234–1242, and histidine-1237 are situated in the nearest neighbourhood to, or directly involved in the formation of the active center of initiating substrate binding of the β subunit of E. coli RNA polymerase.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1989.tb14684.x

Studies on the functional topography of Escherichia coli RNA polymerase. Highly selective affinity labelling by analogues of initiating substrates.

Pt2+ -containing derivatives of oligodeoxyribonucleotides were used to evaluate the ligand affinity to the template sites of Klenow fragment of DNA polymerase I from E. coli and DNA polymerase I fr...

The mechanism of recognition of templates by DNA polymerases from pro- and eukaryotes as revealed by affinity modification data.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/0014-5793%2889%2980439-9

DNA polymerase I (Klenow fragment): Role of the structure and length of a template in enzyme recognition

Interaction of human DNA topoisomerase I with specific sequence oligodeoxynucleotides.

The interaction of EcoRI with different oligodeoxyribonucleotides (ODNs) was analyzed using the method of the slow step-by-step simplification in their complexity. Orthophosphate (KI = 31 mM), 2-deoxyribose 5-phosphate (KI = 4.6 mM) and different dNMPs (KI = 2.1-2.5 mM) were shown to be the minimal ligands of the enzyme. The lengthening of a nonspecific d(pN)n (n = 1-6) by one nucleotide unit resulted in the increase of their affinity by a factor of approximately 2.0. Weak nonspecific electrostatic contacts of EcoRI with internucleotide phosphate groups of ODNs can account for about 5 orders of magnitude in the ligand affinity, whereas the contribution of specific interactions between EcoRI and d(pN)n is no more than 2 orders of magnitude of a total ODN's affinity.

https://iubmb.onlinelibrary.wiley.com/doi/pdf/10.1080/152165401753544269

Tat'yana I. Kolocheva

Papers

Studies on the functional topography of Escherichia coli RNA polymerase. Highly selective affinity labelling by analogues of initiating substrates.

The mechanism of recognition of templates by DNA polymerases from pro- and eukaryotes as revealed by affinity modification data.

DNA polymerase I (Klenow fragment): Role of the structure and length of a template in enzyme recognition

Interaction of human DNA topoisomerase I with specific sequence oligodeoxynucleotides.

Interaction of Endonuclease Eco RI with Short Specific and Nonspecific Oligonucleotides