Journal:

Article Title: Heat shock protein 70 stimulation of the deoxyribonucleic acid base excision repair enzyme polymerase ?

doi:

Figure Lengend Snippet: Heat shock protein 70 (Hsp)70 stimulates β pol repair of pBKS deoxyribonucleic acid after strand scission at AP sites by human apurinic-apyrimidinic endonuclease. (A) Gapped pBKS was radiolabeled during 30 minutes of repair (see Experimental Procedures). Each 12-μL incubation mixture contained 0.150 μg of the phagemid, 2.5 μCi of [α32P]deoxyguanosine triphosphate (dGTP), 1.0 μg of each stimulatory protein, and 1 μL (4 units) of β pol diluted: 1/50 (lanes 5, 10, and 15), 1/100 (lanes 4, 9, and 14), 1/200 (lanes 3, 8, and 13), or 1/400 (lanes 2, 7, and 12). Buffer replaced β pol in treatment groups of lanes 1, 6, and 11. Stimulatory proteins: Hsp70 (lanes 1–5); Hsp70 N-mutant protein (lanes 6–10); Hsp70 C-mutant protein (lanes 11–15). Lane 16 contained 0.100 μg of unlabeled native pBKS. Nicked and covalently closed circular (CCC) forms are shown. CCC signify supercoiled forms. Binding of β pol, [α32P]dGTP, and stimulatory proteins for 30 minutes on ice was followed by addition of pBKS and incubation at 37°C for 30 minutes followed by gel electrophoresis in 0.8% agarose, stained with ethidium bromide and observed under ultraviolet light, as shown in (A). (B) An aligned autoradiogram of the gel in (A) shows incorporation only into the relaxed forms of pBKS; lane numbers correspond to those in (A). The film was exposed for 40 minutes. (C) Densitometer determinations of the autoradiogram of 1B are plotted; the density determination of the N-mutant protein is omitted; •—•, Hsp70; ▵—▵, C-mutant protein. Data from an otherwise similar experiment, in which incorporation at 37°C was for 15 minutes; •—•, Hsp70; ○—○, bovine serum albumin; •—•, no stimulatory protein. The film was exposed for 60 minutes

Article Snippet: The photographic negative was scanned using a Molecular Dynamics Image Quant densitometer (Redwood City, CA, USA), and the proportion of nicked to supercoiled forms was calculated.

Techniques: Incubation, Mutagenesis, Binding Assay, Nucleic Acid Electrophoresis, Staining

Journal:

Article Title: Heat shock protein 70 stimulation of the deoxyribonucleic acid base excision repair enzyme polymerase ?

doi:

Figure Lengend Snippet: Repair of a 30-bp gapped oligonucleotide by β pol and ligases. A gapped 30-bp oligonucleotide containing a single uridine 12 nucleotides from the 5′ terminus of 1 strand was prepared by sequential treatment with uracil–deoxyribonucleic acid (DNA) glycosylase and human apurinic-apyrimidinic endonuclease. Repair of the single-nucleotide gap was achieved by insertion of 32P-labeled deoxycytidine monophosphate. Proteins and unincorporated radiolabel were then eliminated by phenol extraction and ethanol precipitation. Each 20-μL reaction included 6.6 pmol of 32P-labeled oligonucleotide, T4 DNA ligase, and a protein supplement. (A) Aliquots of 3′ 32P-labeled oligonucleotide were incubated at 20°C for 40 minutes with T4 DNA ligase from E coli to seal the 5′ side of the repair site, followed by boiling for 5 minutes in 50% formamide, quick quenching on ice, and electrophoresis on a 16% denaturing acrylamide gel containing 7 M urea. The amounts of T4 DNA ligase shown were supplemented with 0.5 μg of Hsp70, 0.5 μg of bovine serum albumin (BSA), or buffer. The autoradiogram was developed after 2 minutes exposure. (B) A similar study with human DNA ligase 1 is shown. 32P-labeled oligonucleotide (3.3 pmol) was placed in each well. The gel autoradiogram was exposed for 17 minutes. (C) Conversion of 3′ 32P-labeled 12 mers to 30 mers by ligation with T4 DNA ligase and human DNA ligase 1. The autoradiograms of Figure 5 (A and B) were scanned, and densitometer values from labeled 12 mers and repaired 30 mers from each incubation mixture were determined. The proportion of product 30 mers was expressed as a fraction of the total labeled oligonucleotide placed on the gel. With T4 DNA ligase 0.6 pmol was converted; with the human DNA ligase 0.04 pmol of 12 mers was converted during 30 minutes. The amount of T4 DNA ligase or human DNA ligase 1 in each reaction is shown on the abscissa. Hsp70 (0.5 μg) is 7.0 pmol, 1000 ng of T4 DNA ligase is 28 pmol, and 56 ng of human DNA ligase 1 is 5.0 pmol. Human DNA ligase 1, with Hsp70, • human DNA ligase 1 with BSA, ▴; and human DNA ligase 1 with buffer, ▪. T4 DNA ligase with Hsp70, ○; T4 DNA ligase with BSA, ▴; and T4 DNA ligase with buffer, □

Article Snippet: The photographic negative was scanned using a Molecular Dynamics Image Quant densitometer (Redwood City, CA, USA), and the proportion of nicked to supercoiled forms was calculated.

Techniques: Labeling, Extraction, Ethanol Precipitation, Incubation, Electrophoresis, Acrylamide Gel Assay, Ligation