The distribution of DNA repair synthesis in chromatin and its rearrangement following damage with N-acetoxy-2-acetylaminofluorene

Abstract

The distribution of DNA repair synthesis in the chromatin of confluent human diploid fibroblasts damaged with N-acetoxy-2-acetylaminofluorene has been studied. Kinetic analysis of staphylococcal nuclease digestion data revealed that initially most of the repair synthesis occurred in nuclease sensitive regions of chromatin. Continuous labeling experiments and pulse chase experiments indicated that with time much of the 3H dThd initially incorporated into nuclease sensitive regions during repair appeared in nuclease resistant regions. Agarose gel electrophoresis was used to demonstrate that these resistant regions were core DNA. In agreement with previous findings [Smerdon, M.J. and Lieberman, M.W., (1978), Proc. Nat. Acad. Sci. USA, in press], studies of the time course of this rearrangement and of repair synthesis revealed similar time dependences and suggested a relationship between rates of repair synthesis and chromatin rearrangement.

Distribution of DNA damage in chromatin and its relation to repair in human cells treated with 7-bromomethylbenz(a) anthracene

“…2B). All of these data are in agreement with other data (5)(6)(7)(8)24) and help establish the generality of previous findings with respect to "long patch" (UV and UV-mimetic) agents (29). We also find that repair synthesis initiated at times as long as 12 hr after damage still results in the incorporation of nucleotides which are initially SN sensitive (Fig.…”

“…Preparation of nuclei, digestions with staphylococcal nuclease (Worthington), agarose gel electrophoresis, and data analysis were all carried out as described previously (5)(6)(7)(8). Briefly, the analysis of SN digestion kinetics compares the rate of release by SN of damage (3H labeled) or nucleotides incorporated during repair synthesis ( 3H) to nucleotides incorporated into total DNA ( C) in both chromatin (nuclei) and the corresponding purified DNA (5).…”

“…To determine the distribution of repair-incorporated nucleotides immediately following repair synthesis (i.e., before any rearrangement had occurred; Ref. 6,7,8), experimental values of f s/ were extrapolated to "0' time (see Materials and Methods), and a value of 6.2 was obtained. The fraction of the total genome which is SN sensitive (E) was estimated from the H(t) vs C(t) plots (5,8) and found to be 0.17.…”

Cockayne syndrome B protein regulates the transcriptional program after UV irradiation

“…In both cases, CSB has to deal with RNA pol II either when joining the promoter or when stalled in front of a DNA lesion. Whether or not this involves a direct interaction between these two components (Tantin et al , 1997), or induces some chromatin remodelling (Smerdon and Lieberman, 1978; Tlsty and Lieberman, 1978) is unkown. By ChIP and immunoprecipitation assays, we demonstrated that CSB operates at the transcription initiation step to allow recruitment of RNA pol IIA and associated transcription factors on a subset of genes, after UV irradiation.…”

Efficiency of formation of pyrimidine dimers in SV40 chromatin in vitro

“…In order to understand DNA repair in eukaryotic cells, the effect of chromatin structure on DNA damage and repair enzymes must be investigated thoroughly. Repair DNA synthesis is reported to occur preferentially in nuclease-sensitive (noncore) DNA shortly after damage by alkylating agents (Bodell & Banarjee, 1979; Tlsty & Lieberman, 1978;Bodell, 1977;Oleson et al, 1979) or UV radiation (Cleaver, 1977; Williams & Friedberg, 1979;Smerdon & Lieberman, 1978;Smerdon et al, 1979). However, the extent to which repaired nucleotides shift into nuclease-resistant DNA is still unclear.…”