scholarly journals Escherichia coli β-clamp slows down DNA polymerase I dependent nick translation while accelerating ligation

2018 ◽  
Author(s):  
Amit Bhardwaj ◽  
Debarghya Ghose ◽  
Krishan Gopal Thakur ◽  
Dipak Dutta
Keyword(s):  
Escherichia Coli ◽  
Dna Polymerase ◽  
Nick Translation ◽  
Dna Polymerase I ◽  
Okazaki Fragments ◽  
Pol I ◽  
Dna Strands ◽  
Translation Property ◽  
Polymerase I

AbstractThe nick translation property of DNA polymerase I (Pol I) ensures the maturation of Okazaki fragments by removing primer RNAs and facilitating ligation. However, prolonged nick translation traversing downstream DNA is an energy wasting futile process, as Pol I simultaneously polymerizes and depolymerizes at the nick sites utilizing energy-rich dNTPs. Using an in vitro assay system, we demonstrate that the β-clamp of the Escherichia coli replisome strongly inhibits nick translation on the DNA substrate. To do so, β-clamp inhibits the strand displacement activity of Pol I by interfering with the interaction between the finger subdomain of Pol I and the downstream primer-template junction. Conversely, β-clamp stimulates the 5’ exonuclease property of Pol I to cleave single nucleotides or shorter oligonucleotide flaps. This single nucleotide flap removal at high frequency increases the probability of ligation between the upstream and downstream DNA strands at an early phase, terminating nick translation. Besides β-clamp-mediated ligation helps DNA ligase to seal the nick promptly during the maturation of Okazaki fragments.

Caffeine inhibits DNA polymerase I from Escherichia coli: studies in vitro

1982 ◽  
Vol 3 (2) ◽  
pp. 151-153 ◽  
Author(s):  
R. Balachandran ◽  
A. Srinivasan
Keyword(s):  
Escherichia Coli ◽  
Dna Polymerase ◽  
Dna Polymerase I ◽  
Polymerase I

scholarly journals Escherichia coli β-clamp slows down DNA polymerase I dependent nick translation while accelerating ligation

PLoS ONE ◽  
2018 ◽  
Vol 13 (6) ◽  
pp. e0199559 ◽  
Author(s):  
Amit Bhardwaj ◽  
Debarghya Ghose ◽  
Krishan Gopal Thakur ◽  
Dipak Dutta
Keyword(s):  
Escherichia Coli ◽  
Dna Polymerase ◽  
Nick Translation ◽  
Dna Polymerase I ◽  
Polymerase I

Viroid RNA is accepted as a template for in vitro transcription by DNA-dependent DNA polymerase I and RNA polymerase from Escherichia coli

1982 ◽  
Vol 2 (11) ◽  
pp. 929-939 ◽  
Author(s):  
Wolfgang Rohde ◽  
Hans-Richard Rackwitz ◽  
Frank Boege ◽  
Heinz L. Sänger
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Dna Polymerase ◽  
Unit Length ◽  
Dna Polymerase I ◽  
Experimental Conditions ◽  
In Vitro Synthesis ◽  
Polymerase I

The RNA genome of potato spindle tuber viroid (PSTV) is transcribed in vitro into complementary DNA and RNA by DNA-dependent DNA polymerase I and RNA polymerase, respectively, from Escherichia coli. In vitro synthesis of complementary RNA produces distinct transcripts larger than unit length thus reflecting the in vivo mechanism of viroid replication. The influence of varying experimental conditions on the transcription process is studied; actinomycin D is found to drastically reduce complementary RNA synthesis from the PSTV RNA template by RNA polymerase.

scholarly journals Role of Escherichia coli DNA Polymerase I in Conferring Viability upon the dnaN159 Mutant Strain

2007 ◽  
Vol 189 (13) ◽  
pp. 4688-4695 ◽  
Author(s):  
Robert W. Maul ◽  
Laurie H. Sanders ◽  
James B. Lim ◽  
Rosemary Benitez ◽  
Mark D. Sutton
Keyword(s):  
Escherichia Coli ◽  
Dna Polymerase ◽  
Temperature Sensitive ◽  
Mutant Form ◽  
Sliding Clamp ◽  
Pol I ◽  
Pol Iii ◽  
Polymerase I

ABSTRACT The Escherichia coli dnaN159 allele encodes a mutant form of the β-sliding clamp (β159) that is impaired for interaction with the replicative DNA polymerase (Pol), Pol III. In addition, strains bearing the dnaN159 allele require functional Pol I for viability. We have utilized a combination of genetic and biochemical approaches to characterize the role(s) played by Pol I in the dnaN159 strain. Our findings indicate that elevated levels of Pol I partially suppress the temperature-sensitive growth phenotype of the dnaN159 strain. In addition, we demonstrate that the β clamp stimulates the processivity of Pol I in vitro and that β159 is impaired for this activity. The reduced ability of β159 to stimulate Pol I in vitro correlates with our finding that single-stranded DNA (ssDNA) gap repair is impaired in the dnaN159 strain. Taken together, these results suggest that (i) the β clamp-Pol I interaction may be important for proper Pol I function in vivo and (ii) in the absence of Pol I, ssDNA gaps may persist in the dnaN159 strain, leading to lethality of the dnaN159 ΔpolA strain.

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