Completion of excision repair in human cells. Relationship between ligation and nucleosome formation.

M J Smerdon

doi:10.1016/s0021-9258(17)42461-6

Abstract

The completion of excision repair patches in human cells, following UV irradiation, was compared to the refolding of these regions into nucleosomes. Incomplete repair patches were detected by their enhanced sensitivity to exonuclease III. This enhanced sensitivity was due to the presence of gaps (or displaced parental strands) at the 3' end rather than unligated nicks, indicating that ligation occurs rapidly after repair synthesis is completed. Different rates of completion were achieved by treatment with the inhibitors hydroxyurea and sodium butyrate, as well as by using a (partially) ligase-deficient human cell strain. Hydroxyurea caused a marked decrease in both the rate of completion and the level of repair incorporation in all three cell types studied, while sodium butyrate yielded different effects in each cell type. In each case, however, a decrease in the rate of repair patch completion resulted in a concomitant decrease in the level of nucleosome formation. To determine the temporal relationship of these two events, the levels of repair-incorporated nucleotides in isolated 146-base pair nucleosome core DNA were compared on native and denaturing gels. The data indicate that little (or no) nucleosome formation occurred in the nascent DNA regions prior to ligation regardless of the cell type or treatment used. Furthermore, comparison of the fraction of unligated repair patches and the fraction of repair patches in a nonnucleosomal state indicated that in the absence of inhibitors there was a significant time lag between ligation and nucleosome formation. This lag time, however, decreased when cells were treated with hydroxyurea. Thus, the formation of nucleosomes in newly repaired regions of DNA occurred after the ligation step in all cases and these two features of the excision repair process are not "tightly coupled" events.

Highlights

The completion of excision repair patches in human causes a decrease in the rate of nucleosome rearrangement cells, following U V irradiation, was compared to the (Smerdon, 1983)
I n i t ~ l C ~ r ~ t e r i zofaRtewpanir ~ ~ nandtN ~~~ o ss o m~ e Rearrangement in 46BR Celk--I have studied the rates of completion of repair patches and nucleosome rearrangement in newly repaired DNAof normal human fibroblasts from foreskin (AG1518) and fetal lung (IMR go), as well as fibroblasts from an immunodeficient patient which appear to be partially deficient in DNA ligase activity (46BR) (Teo et al, 1983; Squires and Johnson, 1983; Lehmann et al, 1983)
I measuredthe time course of incorporation of [3H]dThdfollowingUV irradiation of confluentcultures of 46BR cells and AG1518cells.As can be seen in Fig. 1, the shape of the [3H]dThd incorporation curve was essentially identical for both cell types during a 24-h periodafter irradiation

Summary

RESULTS

I n i t ~ l C ~ r ~ t e r i zofaRtewpanir ~ ~ nandtN ~~~ o ss o m~ e Rearrangement in 46BR Celk--I have studied the rates of completion of repair patches and nucleosome rearrangement in newly repaired DNAof normal human fibroblasts from foreskin (AG1518) and fetal lung (IMR go), as well as fibroblasts from an immunodeficient patient which appear to be partially deficient in DNA ligase activity (46BR) (Teo et al, 1983; Squires and Johnson, 1983; Lehmann et al, 1983). The rate of loss of the difference between the fraction of 3H rendered acid-soluble enhanced sensitivity decreases in the presence of 2 mM hy- and the corresponding fraction of "C at each digestion time droxyurea (Fig. 2, open circles) These results are similar to If(") - f("C)] was used as a measure of the fraction of those reported previously for normal human fibroblasts incomplete repair patches (Fig. 3, open circles) and denotes (Smerdon and Lieberman, 1980; Smerdon, 1983)and indicate the fraction of exonuclease 111-sensitiverepair sites (ESRS). Ordinate values represent the DNA was prepared from confluent IMR 90cells that were prelabeled ratio f./[ (see "Materials and Methods") determined for the staphy- with ["CIdThd and labeled with [*H]dThd inthe presence of 10mM lococcal nuclease digestion of isolated nuclei (Smerdon et al, 1979).

A G 1 5 1 8 IMR 90 4 6 B R m I

ELECTROPHORESE ON NATIVE AND DENATURING GELS

DISCUSSION