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Replication of DNA in mammalian chromosomes

II. Kinetics of 3H-thymidine incorporation and the isolation and partial characterization of labeled subunits at the growing point

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Abstract

DNA replication has been studied in cells (CHO) synchronized by mitotic selection from roller cultures. A study of the incorporation of 3H supplied as uridine indicates that cells cannot be blocked precisely at the beginning of the S phase, but DNA synthesis can be stopped in early S by treating with F-dU in G1. After blockage potential initiation sites continue to increase at a linear rate for atleast 13 hours after division. Incorporation of 3H-thymidine begins at most of these sites within seconds after thymidine is supplied in the medium and incorporation continues at a linear rate for 20–24 minutes. There appears to be a pause after this interval before synthesis is resumed at about two times the initial rate. 3H-bromodeoxyuridine can be substituted for thymidine without affecting the kinetic pattern over a similar period. The increased rate is probably an increase in sites of chain growth rather than a change in rate of chain growth. A study of the labeled DNA segments by band sedimentation in a preformed NaClO4 isokinetic gradient shows that two distinctly different sized segments can be released from the chromosomes by lysis at submelting conditions. One is the previously reported single chain segments averaging about one-half micron in length, but the other is a much larger segment (26S) which is native DNA with perhaps small regions of single chains presumably at the ends. Primarily single chain DNA is released after 1–2 minute pulse labeling, but after 2 minutes the larger segments (26S) contain most of the newly formed DNA except that attached to the chains of the major part of the template DNA which exhibits a discontinuous distribution, sedimenting far faster than either newly replicated segment. A consideration of the kinetics of formation of the 26S component indicates that is may contain the replicating fork. If this proves to be the correct interpretation the template chains would both have non-adjacent nicks preceeding the fork and also in a post-fork site at a mean distance of about 2 microns in both directions. The isolation of the growing points of DNA replication in chromosomes is now possible and the study of properties of the newly replicated regions should be greatly facilitated.

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Authors and Affiliations

  1. Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida

    J. Herbert Taylor, Alice G. Adams & Michael P. Kurek

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  1. J. Herbert Taylor
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  2. Alice G. Adams
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  3. Michael P. Kurek
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Taylor, J.H., Adams, A.G. & Kurek, M.P. Replication of DNA in mammalian chromosomes. Chromosoma 41, 361–384 (1973). https://doi.org/10.1007/BF00396495

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