DNA complementary to rabbit globin mRNA made by E. coli polymerase I

Incubation of liver microsomes with cytochrome $\text{b}{}_5$, purified after solubilization with detergents, caused an effective incorporation of the cytochrome into the microsomal membranes. The incorporated cytochrome was reducible by NADH and could not be removed by repeated washing with 0.3 M KCl or 10 mM EDTA. The incorporation was much more efficient at 37°C than at 0°C. Trypsin-solubilized cytochrome $\text{b}{}_5$, which lacks the hydrophobic tail of the native protein, could not be inserted into the membranes. These findings confirm the view that the hydrophobic tail of the cytochrome molecule is responsible for its tight binding to the microsomal membranes.     

Nucleotide excision by E. coli DNA polymerase I in proofreading and non-proofreading modes

Escherichia coli DNA polymerase I exists in at least two distinct kinetic forms. When it binds to a template, the proofreading activity is usually switched off. As the enzyme progresses along the template, it becomes more and more competent for excision. This phenomenon introduces a link between fidelity and processivity. Processivity is best studied when the chain-length distributions of synthesized polymers are stationary. Even then, however, one cannot avoid multiple initiations on a given template by the same molecule of the enzyme. When synthesis is initiated with primers of lengths 15 or 20, a strange phenomenon is observed. It seems that the polymerase starts by hydrolyzing the primer down to a length of 7-10 nucleotides and only then starts to add nucleotides. It does so in a low-accuracy mode, suggesting that, while the exonuclease is clearly active, it does not contribute to proofreading. The warm-up of the proofreading function is therefore reinterpreted as a switch between two modes of behaviour: a mode 1 of low accuracy in which the 3'----5' exonuclease, while active, is uncoupled from the polymerase and does not contribute to proofreading, and a mode 2 of high accuracy in which the exonuclease is kinetically linked to the polymerase activity.     

Excision of thymine dimers by proteolytic and amber fragments of E. coli DNA polymerase I

Excision of thymine dimers from specifically incised ultraviolet irradiated DNA by $\text{E. coli}$ DNA polymerase I is stimulated by concurrent DNA synthesis. The 36,000 molecular-weight “small fragment” obtained by limited proteolysis of DNA polymerase I, which retains only the 5′ → 3′ exonuclease activity, also excises thymine dimers, but at one-tenth the rate of the intact enzyme. However, the rate of excision is increased by addition of the “large” 76,000-molecular weight fragment. With the further addition of the 4 deoxynucleoside triphosphates, permitting DNA synthesis to occur, excision approaches rates observed with the intact enzyme. The same result was obtained with a fragment of DNA polymerase I with 5′ → 3′ exonuclease activity that is present uniquely in polymerase I $\text{amber}$ mutants.     

Methylation-dependent DNA synthesis in Escherichia coli mediated by DNA polymerase I.

An in vitro system was used to study DNA synthesis in lysates of Escherichia coli cells which had been grown in the presence of ethionine. Such lysates showed a reduced capacity to incorporate [3H]TTP into high-molecular-weight material. Activity could be restored by incubation with S-adenosyl methionine and ATP. S-adenosyl methionine-reactivated TTP incorporation required the presence of DNA polymerase I, ATP, and all four deoxyribonucleotide triphosphates. DNA polymerase III was not required.     

Poly(dG-dC) in the Z-form inhibits E. coli DNA polymerase I and AMV DNA polymerase activity

The effect of Z-conformation of DNA on its template activity in DNA synthesis reactions in vitro has been studied. Normal poly(dG-dC) in the B-form, brominated and unbrominated in the Z-form have been compared for their template activity in DNA synthesis reactions mediated by AMV DNA polymerase and E. coli DNA polymerase I. The results indicate that poly(dG-dC) in the Z-form is totally inactive as a template for DNA synthesis and further that it is a strong competitive inhibitor of copying of the B-form DNA.     

Sequence specificity of DNA cleavage by bis(1,10-phenanthroline)copper(I)

The bis(1,10-phenanthroline)copper(I) complex is a relatively simple molecule previously shown to cause DNA cleavage with a strong preference for gene control regions such as the Pribnow box. Sequence level mapping of sites of [(Phen)2CuI]+ cleavage in greater than 2000 bases in histone genes and the plasmid pUC9 showed that the specificity for control regions is related to a predominant preference for minor groove binding at TAT triplets, which were cleaved most strongly at the adenosine sugar ring. The related sequences TGT, TAAT, TAGPy, and CAGT (Py = pyrimidine) were moderately preferred, while CAT and TAC triplets, PyPuPuPu quartets, PuPuPuPy quartets, and CG-rich PyPuPuPy quartets were cleaved with low to average frequency. Polypurine and polypyrimidine sequences were cleaved with low frequency. The sequence preferences of [(Phen)2CuI]+ can be ascribed predominantly to (i) a requirement for binding in the minor groove at a pyrimidine 3'----5' step and (ii) stereoelectronic effects of the 2-amino group of guanine in the minor groove, which inhibit binding. Although the reagent appears primarily to recognize sequence features at the triplet or quartet level, lower than expected cleavage was observed for two TAT sequences adjacent to several other preferred sequences and higher than expected cleavage was observed at CAAGC sequences, suggesting that longer range sequence-dependent DNA conformational effects influence specificity in certain cases.