Human DNA ligase I efficiently seals nicks in nucleosomes - PubMed (original) (raw)
Human DNA ligase I efficiently seals nicks in nucleosomes
D R Chafin et al. EMBO J. 2000.
Abstract
The access to DNA within nucleosomes is greatly restricted for most enzymes and trans-acting factors that bind DNA. We report here that human DNA ligase I, which carries out the final step of Okazaki fragment processing and of many DNA repair pathways, can access DNA that is wrapped about the surface of a nucleosome in vitro and carry out its enzymatic function with high efficiency. In addition, we find that ligase activity is not affected by the binding of linker histone (H1) but is greatly influenced by the disposition of the core histone tail domains. These results suggest that the window of opportunity for human DNA ligase I may extend well beyond the first stages of chromatin reassembly after DNA replication or repair.
Figures
Fig. 1. Nucleosomal DNA substrates for human DNA ligase I. Three double-stranded substrates for human DNA ligase I were constructed as described in Materials and methods. Each contains the same NPE with the expected position of the nucleosome indicated by the oval. The 154 bp substrate contains a single nick near the predicted location of the nucleosome dyad. The 218 bp substrates consist of the 154 bp sequence with 64 bp appended to the right-hand end and either contains a single nick near the nucleosome dyad or three nicks at the indicated locations. The length of oligonucleotides used to assemble the substrates is indicated. The positions of radioactive phosphate labels are indicated (circles). Identical DNAs lacking nicks were also constructed (not shown; see Figure 2).
Fig. 2. Hydroxyl radical characterization of nucleosomal ligase substrates. Nucleosomes (Nuc) and free DNA (FD) were reacted with hydroxyl radicals (⋅OH), isolated, and then cleavage patterns analyzed on 6% sequencing gels as described in Materials and methods. Shown are reactions for the 218 (left) or 154 bp (right) intact double-stranded fragments (lanes 1–3) or nicked substrates (lanes 4–6). Lane G contains products of the Maxam–Gilbert G-specific sequencing reaction for each DNA. Lanes 1 and 6 in each gel contain free DNA not reacted with hydroxyl radicals while products from cleavage of naked DNA or nucleosomes are shown in lanes 2 and 5 or lanes 3 and 4, respectively. A schematic depicting the 5S DNA fragment and the position of the nucleosome (oval) based on the footprinting results is shown. The locations of nicks when present on the opposite strand are indicated (circles).
Fig. 3. Efficient ligation of a nick in a nucleosome. Nucleosomes were reconstituted with the 218 bp substrate containing three nicks and treated with human DNA ligase I as described in Materials and methods. Labeled reactants and products from the nucleosome (Nuc) or the free DNA (FD) were isolated from a 0.7% agarose gel and analyzed on a 6% sequencing gel. Lanes 1–5 and 6–10 contain ligation products from free DNA or nucleosomes generated after 0, 5, 10, 30 and 60 min, respectively. Oligonucleotides in the original substrate are shown in the schematic below the gel and the products of ligation at each of the three labeled sites (circles, schematic) are indicated alongside the gel.
Fig. 4. Rate of ligation within the 218 bp nucleosome substrate is not affected by nucleosome translational heterogeneity or binding of linker histone H1. (A) Isolation of nucleosome translational isomers. Nucleosomes were assembled with the 218 bp template containing a single nick (Figure 1), subjected to human DNA ligase I, and labeled ligation products were isolated on a 5% polyacrylamide ‘translational’ gel. Lanes 1–8 contain nucleosomes treated with 15 U of human DNA ligase I for 0.25, 0.5, 1, 2, 4, 8, 16 and 32 min. Schematics of nucleosome positions for each translational isomer as determined by hydroxyl radical footprinting are shown (right). (B) Characterization of ligation within individual nucleosome translational isomers. Labeled DNA was isolated from nucleosome and naked DNA bands as shown in (A) and ligation products analyzed on 6% sequencing gels as in Figure 3. Free DNA (lanes 1–8) and nucleosome samples (lanes 9–24) were obtained from reactions containing 1 or 15 U of ligase, respectively. Lanes 9–16 or 17–24 show results for the ‘upper’ or ‘lower’ nucleosomes from (A). (C) Binding of linker histone H1 to nucleosome substrates does not affect the efficiency of ligase. The 218 bp nucleosome substrate containing three nicks was prepared and incubated in the presence or absence of H1 before the addition of 1 U of ligase for 10 min. Reactions were loaded onto nucleoprotein gels to isolate linker histone-bound and/or unbound nucleosomes before sequencing gel analysis of ligase products. Lane 1, no ligase; lanes 2, 3 and 4, ligation within free DNA, nucleosomes or nucleosomes bound by linker histone, respectively. Bands are as indicated in Figure 3.
Fig. 5. Ligation of 154 bp nucleosome core substrates. Nucleosome cores were assembled with the 154 bp substrate (Figure 1), then subjected to the indicated levels of human DNA ligase for the indicated times before separation of free DNA and nucleosomes on preparative nucleoprotein gels and analysis of ligation on 6% sequencing gels. Lane 1, no ligase added; lane 2, ligation within free DNA; lanes 3 and 4, ligation of nucleosomal DNA containing full-length histones (WT) with 1 U of ligase for 1 min or with 50 U of ligase for 30 min, respectively; lane 5, ligation within trypsinized 154 bp nucleosomes lacking core histone tail domains (Try). Positions of 88 nucleotide labeled unligated oligonucleotide and 154 bp ligation product are indicated to the right of the gel.
Fig. 6. Removal of histone tail domains does not substantially increase site exposure within the nucleosome. (A) Schematic of nucleosomes. Positions of nucleosome (oval) and _Sac_I sites (block) are indicated. Except for the introduction of the _Sac_I site (see text), templates are identical to those shown in Figure 1. (B) _Sac_I digestion of nucleosomes and naked DNA. Nucleosomes reconstituted with the 154 bp substrate were mixed with the 218 bp free DNA and subjected to either 4 U/ml (not shown) or 10 000 U/ml (shown) of _Sac_I restriction endonuclease for various periods of time, then digestion products analyzed on non-denaturing acrylamide gels. Digestion products from wt or trypsinized nucleosomes are shown. Samples in lanes 1–9 were digested for 0, 0.5, 5, 15, 30, 45, 60, 90 and 120 min, respectively. Note that _Sac_I cleavage of both the nucleosomal template and the naked DNA control yields the same size DNA product (Frags). (C) Kinetics of _Sac_I digestion of nucleosomes and naked DNA. Data such as those shown in (B) were quantitated and plotted as the log fraction of uncut DNA remaining versus time of digestion. Shown are plots from digestions of 154 bp nucleosomes containing wt or trypsinized (tailless) nucleosomes with 10 000 U/ml _Sac_I and naked DNA digested with 4 U/ml _Sac_I, as indicated in the inset. Lines represent the results of fits of a single exponential first-order decay to the data.
Fig. 7. Accessibility of human DNA ligase I during chromatin assembly after passage of the replication fork. Structures predicted to be relatively accessible or inaccessible to ligase I are indicated.
References
- Anderson J.D. and Widom,J. (2000) Sequence and position-dependence of the equilibrium accessibility of nucleosomal DNA target sites. J. Mol. Biol., 296, 979–987. - PubMed
- Anderson S. and DePamphilis,M.L. (1979) Metabolism of Okazaki fragments during simian virus 40 DNA replication. J. Biol. Chem., 254, 11495–11504. - PubMed
- Annunziato A.T. and Seale,R.L. (1982) Maturation of nucleosomal and non-nucleosomal components of nascent chromatin: differential requirements for concurrent protein synthesis. Biochemistry, 21, 5431–5438. - PubMed
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