Vincent J. Cannistraro

Rotational position of a 5-methylcytosine-containing cyclobutane pyrimidine dimer in a nucleosome greatly affects its deamination rate.

C to T mutation hotspots in skin cancers occur primarily at methylated CpG sites that coincide with sites of UV-induced cyclobutane pyrimidine dimer (CPD) formation. These mutations are proposed to arise from the insertion of A by DNA polymerase η opposite the T that results from deamination of the methylC (mC) within the CPD. Although the frequency of CPD formation and repair is modestly modulated by its rotational position within a nucleosome, the effect of position on the rate of mC deamination in a CPD has not been previously studied. We now report that deamination of a TmC CPD whose sugar phosphate backbone is positioned against the histone core surface decreases by a factor of 4.7, whereas that of a TmC CPD positioned away from the surface increases by a factor of 8.9 when compared with unbound DNA. Because the mCs undergoing deamination are in similar steric environments, the difference in rate appears to be a consequence of a difference in the flexibility and compression of the two sites due to DNA bending. Considering that formation of the CPD positioned away from the surface is also enhanced by a factor of two, a TmCG site in this position might be expected to have up to an 84-fold higher probability of resulting in a UV-induced mC to T mutation than one positioned against the surface. These results indicate that rotational position may play an important role in the formation of UV-induced C to T mutation hotspots, as well as in the mutagenic mechanism of other DNA lesions.

Synergistic modulation of cyclobutane pyrimidine dimer photoproduct formation and deamination at a TmCG site over a full helical DNA turn in a nucleosome core particle

Sunlight-induced C to T mutation hotspots in skin cancers occur primarily at methylated CpG sites that coincide with sites of UV-induced cyclobutane pyrimidine dimer (CPD) formation. The C or 5-methyl-C in CPDs are not stable and deaminate to U and T, respectively, which leads to the insertion of A by DNA polymerase η and defines a probable mechanism for the origin of UV-induced C to T mutations. We have now determined the photoproduct formation and deamination rates for 10 consecutive T=mCG CPDs over a full helical turn at the dyad axis of a nucleosome and find that whereas photoproduct formation and deamination is greatly inhibited for the CPDs closest to the histone surface, it is greatly enhanced for the outermost CPDs. Replacing the G in a T=mCG CPD with A greatly decreased the deamination rate. These results show that rotational position and flanking sequence in a nucleosome can significantly and synergistically modulate CPD formation and deamination that contribute to C to T mutations associated with skin cancer induction and may have influenced the evolution of the human genome.

Ability of Polymerase η and T7 DNA Polymerase to Bypass Bulge Structures

DNA misalignment occurs in homopolymer tracts during replication and can lead to frameshift mutations. Polymerase (pol) recognition of primer-templates containing bulge structures and the transmission of a bulge through a polymerase binding site or replication complex are important components of frameshift mutagenesis. In this report, we describe the interaction of the catalytic core of pol η with primer-templates containing bulge structures by single round primer extension. We found that pol η could stabilize a frayed primer terminus, which enhances its ability to extend primer-templates containing bulges. Based on methylphosphonate-DNA mapping, pol η interacts with the single strand template but not appreciably with the template strand of the DNA stem greater than two nucleotides from the primer terminus. These latter characteristics, combined with the ability to stabilize a frayed primer terminus, may explain why primer-templates containing template bulges are extended so efficiently by pol η. Although pol η could accommodate large bulges and continue synthesis without obstruction, bulge structures in the template, but not in the primer, caused termination of the T7 DNA replication complex. Terminations occurred when the template bulge neared the helix-loop-helix domain of the polymerase thumb. Terminations were not observed, however, when bulge structures approached the site of interaction of the DNA with the extended thumb and thioredoxin. At low temperature, however, terminations did occur at this site.

Rapid deamination of cyclobutane pyrimidine dimer photoproducts at TCG sites in a translationally and rotationally positioned nucleosome in vivo.

Background: DNA photoproduct deamination contributes to mutations. Results: Cyclobutane pyrimidine dimers (CPDs) deaminate fastest at TCG sites, and the rate depends on the position of the CPD in a nucleosome determined from hydroxyl radical footprinting. Conclusion: Deamination could explain the high C to T mutation rate at TCG sites, which can be further modulated by nucleosomes. Significance: Sequence context and chromatin structure can modulate UV mutagenesis. Sunlight-induced C to T mutation hot spots in skin cancers occur primarily at methylated CpG sites that coincide with sites of UV-induced cyclobutane pyrimidine dimer (CPD) formation. The C and 5-methyl-C in CPDs are not stable and deaminate to U and T, respectively, which leads to the insertion of A by the DNA damage bypass polymerase η, thereby defining a probable mechanism for the origin of UV-induced C to T mutations. Deamination rates for TmCG CPDs have been found to vary 12-fold with rotational position in a nucleosome in vitro. To determine the influence of nucleosome structure on deamination rates in vivo, we determined the deamination rates of CPDs at TCG sites in a stably positioned nucleosome within the FOS promoter in HeLa cells. A procedure for in vivo hydroxyl radical footprinting with Fe-EDTA was developed, and, together with results from a cytosine methylation protection assay, we determined the translational and rotational positions of the TCG sites. Consistent with the in vitro observations, deamination was slower for one CPD located at an intermediate rotational position compared with two other sites located at outside positions, and all were much faster than for CPDs at non-TCG sites. Photoproduct formation was also highly suppressed at one site, possibly due to its interaction with a histone tail. Thus, it was shown that CPDs of TCG sites deaminate the fastest in vivo and that nucleosomes can modulate both their formation and deamination, which could contribute to the UV mutation hot spots and cold spots.

Methyl CpG binding protein 2 (MeCP2) enhances photodimer formation at methyl-CpG sites but suppresses dimer deamination

Spontaneous deamination of cytosine to uracil in DNA is a ubiquitous source of C→T mutations, but occurs with a half life of ∼50 000 years. In contrast, cytosine within sunlight induced cyclobutane dipyrimidine dimers (CPDs), deaminate within hours to days. Methylation of C increases the frequency of CPD formation at PyCG sites which correlate with C→T mutation hotspots in skin cancers. MeCP2 binds to mCG sites and acts as a transcriptional regulator and chromatin modifier affecting thousands of genes, but its effect on CPD formation and deamination is unknown. We report that the methyl CpG binding domain of MeCP2 (MBD) greatly enhances C=mC CPD formation at a TCmCG site in duplex DNA and binds with equal or better affinity to the CPD-containing duplex compared with the undamaged duplex. In comparison, MBD does not enhance T=mC CPD formation at a TTmCG site, but instead increases CPD formation at the adjacent TT site. MBD was also found to completely suppress deamination of the T=mCG CPD, suggesting that MeCP2 may have the capability to both suppress UV mutagenesis at PymCpG sites as well as enhance it.

Isolating and sequencing the predominant 5'-ends of a specific mRNA in cells. I. Purification by filter hybridization.

Procedures are considered for purification of a specific procaryotic RNA by successive hybridizations to DNA immobilized to nitrocellulose with special consideration of problems associated with subsequent end-labeling in the T4 polynucleotide kinase reaction. (1) Inhibitors of the kinase can be associated with the plasmid but were removed by electrophoresis of the DNA fragment through polyacrylamide. (2) Residual soluble acrylamide, contaminating the DNA and preventing its efficient retention to nitrocellulose, could be removed by DE52 chromatography. (3) Short denatured DNA required high salt (0.9 M) to bind to nitrocellulose but reannealed quickly at those salt concentrations unless applied at less than or equal to 0.3 micrograms/ml at 4 degrees C with a flow rate of 1 ml/min. (4) The kinetics of the hybrid reaction were a function of DNA length, concentration, and temperature. (5) Formamide was a more effective denaturing agent to remove hybrid RNA from the filter than either 12 M urea or 8 M guanidine-HCl, but caused significant release of DNA from the nitrocellulose as well as another potent inhibitor of the kinase reaction. The release of DNA and other kinase inhibitors was greatly reduced by eluting in boiling water.