On the molecular weight and subunit composition of calf thymus ribonuclease H1

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Abstract
We have reinvestigated the molecular weight and subunit composition of calf thymus ribonuclease H1. Earlier studies suggested a variety of molecular weights for the enzyme in the range of 64K-84K and reported that the enzyme either was a single polypeptide of 74 kDa or consisted of from two to four subunits in the range of 21-34 kDa. Although we too find bands in this lower molecular weight range in our highly purified preparations following SDS-PAGE, our data suggest that the native structure of RNase H1 is a dimer of 68-kDa subunits. The evidence includes the following: (1) Western blot analysis of fractions taken at various stages of the purification indicates that the predominant antigenic form of the enzyme in crude extracts has a molecular weight of 68K but that during purification in the absence of sufficient protease inhibitors a variety of lower molecular weight forms appear concomitant with the disappearance of the 68-kDa band. (2) Activity gel analysis of the highly purified enzyme prepared in the presence of a battery of protease inhibitors reveals that the 8-kDa band (as well as several bands of lower molecular weight) possesses RNase H activity. (3) The 68-kDa band recognized by Western blotting with anti-RNase H immune sera is not detected by using preimmune sera. Furthermore, when immune sera are used, a trace of a 140-150-kDa antigenic form can sometimes be detected, consistent with the existence of a dimeric form of the enzyme. (4) Gel filtration analysis of highly purified RNase H1 reveals that nearly all the observed RNase H activity is found at an elution volume corresponding to a molecular weight in the range of 140K-150K, although under the conditions of sucrose gradient centrifugation, the majority of the enzyme appears to sediment as a 68-kDa monomer. We therefore suggest that the proteolytic lability of RNase H1 may account for earlier reports that the enzyme contains low molecular weight subunits and that the enzyme is composed of a 68-kDa polypeptide which can dimerize and remain active.