DNA Polymerase Inhibition Assay (PIA) for the Detection of Drug–DNA Interactions

2003 ◽  
pp. 117-126
Author(s):  
Thomas P. Dooley ◽  
Katherine L. Weiland
Keyword(s):  
Dna Polymerase ◽  
Inhibition Assay ◽  
Dna Interactions

Micromethod for phosphonoformate inhibition assay of hepatitis B viral DNA polymerase.

1984 ◽  
Vol 30 (4) ◽  
pp. 549-552 ◽  
Author(s):  
H J Lin ◽  
P C Wu ◽  
C L Lai ◽  
W Chak
Keyword(s):  
Hepatitis B ◽  
Dna Polymerase ◽  
Specific Activity ◽  
Sample Volume ◽  
Inhibition Assay ◽  
Viral Dna ◽  
Good Discrimination ◽  
Human Dna ◽  
Nucleotide Incorporation ◽  
B Virus

Abstract A micromethod for the specific measurement of hepatitis B viral DNA polymerase in serum is presented, based on the phosphonoformate inhibition assay (J Med Virol 12: 61-70, 1983). In the micromethod, sample volume is reduced to 120 microL and the ultracentrifugation step is eliminated. The method allows good discrimination between serum infected with hepatitis B virus and uninfected serum. The cutoff value for rate of nucleotide incorporation, based on assays of 41 serum specimens negative for hepatitis B serological markers, was about 15 nU/L (90th percentile). Serum containing hepatitis B surface and antigens exhibited rates of phosphonoformate-inhibitive nucleotide incorporation of 150 (SD 150) nU/L, with an upper 90th percentile range of 17 to 667 nU/L (n = 41). The micromethod makes use of commercially available [32P]dCTP (specific activity about 7000 kCi/mol). 125I-labeled dCTP was found to be unsuitable for this assay. Human DNA polymerases in serum are detected by this method but are excluded from the phosphonoformate-inhibitive fraction.

scholarly journals Stepwise assembly of the human replicative polymerase holoenzyme

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Mark Hedglin ◽  
Senthil K Perumal ◽  
Zhenxin Hu ◽  
Stephen Benkovic
Keyword(s):  
Dna Polymerase ◽  
Clamp Loader ◽  
Dna Interactions ◽  
Sliding Clamp ◽  
Stepwise Manner ◽  
Protein Dna Interactions ◽  
Clamp Loaders ◽  
Sliding Clamps ◽  
Stepwise Assembly

In most organisms, clamp loaders catalyze both the loading of sliding clamps onto DNA and their removal. How these opposing activities are regulated during assembly of the DNA polymerase holoenzyme remains unknown. By utilizing FRET to monitor protein-DNA interactions, we examined assembly of the human holoenzyme. The results indicate that assembly proceeds in a stepwise manner. The clamp loader (RFC) loads a sliding clamp (PCNA) onto a primer/template junction but remains transiently bound to the DNA. Unable to slide away, PCNA re-engages with RFC and is unloaded. In the presence of polymerase (polδ), loaded PCNA is captured from DNA-bound RFC which subsequently dissociates, leaving behind the holoenzyme. These studies suggest that the unloading activity of RFC maximizes the utilization of PCNA by inhibiting the build-up of free PCNA on DNA in the absence of polymerase and recycling limited PCNA to keep up with ongoing replication.

scholarly journals Enzyme-DNA Interactions Required for Efficient Nucleotide Incorporation and Discrimination in Human DNA Polymerase β

1996 ◽  
Vol 271 (21) ◽  
pp. 12141-12144 ◽  
Author(s):  
William A. Beard ◽  
Wendy P. Osheroff ◽  
Rajendra Prasad ◽  
Michael R. Sawaya ◽  
Madhuri Jaju ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Dna Interactions ◽  
Dna Polymerase Β ◽  
Human Dna ◽  
Nucleotide Incorporation

scholarly journals Sliding Clamp–DNA Interactions Are Required for Viability and Contribute to DNA Polymerase Management in Escherichia coli

2009 ◽  
Vol 387 (1) ◽  
pp. 74-91 ◽  
Author(s):  
Justin M.H. Heltzel ◽  
Sarah K. Scouten Ponticelli ◽  
Laurie H. Sanders ◽  
Jill M. Duzen ◽  
Vivian Cody ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Polymerase ◽  
Dna Interactions ◽  
Sliding Clamp

scholarly journals Single Molecule DNA Interactions Between the E. Coli DNA Polymerase III α Subunit and the Polymerase Manager Protein UmuD

2012 ◽  
Vol 102 (3) ◽  
pp. 280a-281a
Author(s):  
Clarissa Ruslie ◽  
Kathy Chaurasiya ◽  
Michelle C. Silva ◽  
Philip Nevin ◽  
Penny J. Beuning ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Single Molecule ◽  
Dna Polymerase Iii ◽  
Dna Interactions ◽  
Α Subunit ◽  
E Coli ◽  
Polymerase Iii
Sign in / Sign up

Export Citation Format

Share Document