Abstract
A procedure is described for the purification from cultured mouse cells of two DNA polymerase "delta-like" enzymes, as defined by intrinsic 3'-exonuclease activity, inhibition by aphidicolin, and relative insensitivity to N2-(p-n-butylphenyl)-dGTP. One of the two enzymes has been purified to near homogeneity and, similar to the DNA polymerase delta from calf thymus described by Lee et al. (Lee, M. Y. W. T., Tan, C. K., Downey, K. M., and So, A. G. (1984) Biochemistry 23, 1906-1913), it has a total molecular mass of 178 kDa (from sedimentation velocity of 8.0 S and Stokes radius of 54 A) and is composed of one each of 125- and 50-kDa polypeptides. It also resembles the DNA polymerase delta of Lee et al. in being stimulated by proliferating cell nuclear antigen (PCNA). It is the first clear structural and functional counterpart of the calf thymus enzyme. The major difference between the mouse DNA polymerase delta and the calf thymus enzyme of Lee et al. is that, under specific conditions, the mouse enzyme is active with poly(dA).oligo(dT) in the absence of PCNA, whereas the activity of the calf thymus enzyme with this template is reported to be completely dependent on PCNA. The reason for this difference is not known at this time. The second mouse cell enzyme has a molecular mass of 112 kDa (from sedimentation velocity of 6.3 S and Stokes radius of 43.0 A) and consists of a single polypeptide of 123-125 kDa in denaturing gels (p125). On the basis of its apparent formation by dissociation of DNA polymerase delta, and multiple similarities with DNA polymerase delta in enzymatic properties, the p125 is provisionally identified as the 125-kDa polypeptide of DNA polymerase delta. The p125 does not respond to PCNA, suggesting that the 50-kDa polypeptide is required for the stimulation of DNA polymerase delta by PCNA. The presence of the p125 in cell extracts would explain reports that DNA polymerase delta consists of a single polypeptide of approximately 125 kDa and/or thast it has a smaller molecular mass than DNA polymerase delta of Lee et al. and is not affected by PCNA (this does not apply to PCNA-independent DNA polymerase delta-like enzymes with higher molecular mass than the polymerase delta of Lee et al., which have recently been named DNA polymerases epsilon).
Highlights
We describe here the purification from mouse cells of two enzymes that have properties of DNA polymerase 6, the larger of which conforms to the structure of the calf thymus enzyme of Lee et al and its sensitivity to proliferating cell nuclear antigen (PCNA)
Subunit structure, intrinsic exonuclease activity, pattern of responses to inhibition, and stimulation by PCNA (2-5, lo), we conclude that the larger enzyme described here is the mouse counterpart of the calf thymus DNA polymerase 6 described by Lee et al (2)
Since the latter report there have been several detailed descriptions of enzymes that have an intrinsic 3’-exonuclease activity and respond to inhibitors as expected for DNA polymerase 6; subunit sizes and native molecular masses of these have been larger than the enzyme of Lee et al, and they were not stimulated by PCNA (11-14)
Summary
Reagents-DNA polymerase a/primase was purified from mouse cells as described previously (20). With 40 g of cells), was -90% pure by Assays-The assay for DNA polymerase 6 activity throughout the purification was based on procedures of Lee et al (2) It was carried out in 20 ~1 containing 50 mM Bis-Tris(Cl-), pH 6.5, 5 mM MgCl,, 100 @M dATP, 20 PM [“H]dTTP (5 &i/nmol), and 15 pg/ml of DNase-treated poly(dA-dT). 37 “C, following which acid-insoluble radioactivity was measured These conditions appeared to give higher activities during intermediate steps in the purification but were not optimal for the purified d polymerases After 30 min at 37 “C!, 10 bl was added of a mixture containing 0.1 M EDTA, 3 mg/ml herring sperm DNA, 10 mg/ml bovine plasma albumin, and 1 mM rUMP, followed by 2 ~1 of trichloroacetic acid (1 g/ml). ’ Proteins in this step were estimated from silver-stained gels
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
