Study on a hidden protein-DNA binding in salmon sperm DNA sample by dynamic kinetic capillary isoelectric focusing.
Nuclease P1 is an important enzyme that hydrolyzes RNA or single-stranded DNA into nucleotides, and complete digestion is an essential basis for assays based on this enzyme. To digest a doubled-stranded DNA, the enzyme is usually combined with heat denaturing, which breaks doubled-stranded DNA into single strands. This paper presents an un-expected phenomenon that nuclease P1, in combination with heat denaturing, fails to completely digest a DNA sample extracted from salmon sperm. Under the experimental conditions used, at which nuclease P1 can completely digest calf thymus DNA, the digestion yield of salmon sperm DNA was only 89.5%. Spectrometric measurement indicated that a total protein of 4.7% is present in the DNA sample. To explain the reason for this phenomenon, the dynamic kinetic capillary isoelectric focusing (DK-CIEF) approach proposed previously, which allows for the discrimination of different types of protein-DNA interactions and the measurement of the individual dissociation rate constants, was modified and applied to examine possible protein-DNA interactions involved. It was found that a non-specific DNA-protein binding occurs in the sample, the dissociation rate constant for which was measured to be 7.05+/-0.83x10(-3) s(-1). The formation of DNA-protein complex was suggested to be the main reason for the incomplete digestion of the DNA sample. The modified DK-CIEF approach can be applied as general DNA samples, with the advantages of fast speed and low sample consumption.
Liang
Liang
L
School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China.
Dou
Peng
P
Dong
Mingming
M
Ke
Xiaokang
X
Bian
Ningsheng
N
Liu
Zhen
Z
D000818:Animals; D004247:DNA / Q000032:analysis* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000032:analysis; D019075:Electrophoresis, Capillary / Q000379:methods*; D007525:Isoelectric Focusing / Q000379:methods*; D007700:Kinetics; D008297:Male; D011485:Protein Binding; D012474:Salmon / Q000235:genetics*; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism; D013094:Spermatozoa / Q000737:chemistry
D004268:DNA-Binding Proteins; D004247:DNA; D015719:Single-Strand Specific DNA and RNA Endonucleases