Eder P S, Walder J A
Department of Biochemistry, University of Iowa, Iowa City 52242.
J Biol Chem. 1991 Apr 5;266(10):6472-9.
The major ribonuclease H from K562 human erythroleukemia cells has been purified more than 4,000-fold. This RNase H, now termed RNase H1, is an endoribonuclease whose products contain 5'-phosphoryl and 3'-hydroxyl termini. The enzyme has a native molecular weight of 89,000 based on its sedimentation and diffusion coefficients. Human RNase H1 has an absolute requirement for a divalent cation. Maximal activity is obtained with either 10 mM Mg2+, 5 mM Co2+, or 0.5 mM Mn2+. The pH optimum is between 8.0 and 8.5 in the presence of 10 mM Mg2+. The isoelectric point is 6.4. RNase H1 lacks double-stranded and single-stranded RNase and DNase activities, and it will not hydrolyze the DNA moiety of an RNA.DNA heteroduplex. Unlike the Escherichia coli enzyme, which requires a heteroduplex that contains at least four consecutive ribonucleotides for activity, human RNase H1 can hydrolyze a DNA.RNA.DNA/DNA heteroduplex that contains a single ribonucleotide. Cleavage occurs at the 5' phosphodiester of this residue. This substrate specificity suggests that human RNase H1 could play a role in ribonucleotide excision from genomic DNA during replication.
来自人红白血病K562细胞的主要核糖核酸酶H已被纯化了4000多倍。这种核糖核酸酶H，现称为核糖核酸酶H1，是一种内切核糖核酸酶，其产物含有5'-磷酸和3'-羟基末端。根据其沉降和扩散系数，该酶的天然分子量为89,000。人核糖核酸酶H1绝对需要二价阳离子。在10 mM Mg2+、5 mM Co2+或0.5 mM Mn2+存在下可获得最大活性。在10 mM Mg2+存在下，最适pH值在8.0至8.5之间。等电点为6.4。核糖核酸酶H1缺乏双链和单链核糖核酸酶及脱氧核糖核酸酶活性，并且它不会水解RNA.DNA异源双链体的DNA部分。与大肠杆菌酶不同，后者需要含有至少四个连续核糖核苷酸的异源双链体才能发挥活性，人核糖核酸酶H1可以水解含有单个核糖核苷酸的DNA.RNA.DNA/DNA异源双链体。切割发生在该残基的5'磷酸二酯处。这种底物特异性表明人核糖核酸酶H1可能在复制过程中从基因组DNA切除核糖核苷酸中发挥作用。
