Nucleosomes suppress the formation of double-strand DNA breaks during attempted base excision repair of clustered oxidative damages.

Exposure to ionizing radiation can produce multiple, clustered oxidative lesions in DNA. The near simultaneous excision of nearby lesions in opposing DNA strands by the base excision repair (BER) enzymes can produce double-strand DNA breaks (DSBs). This attempted BER accounts for many of the potentially lethal or mutagenic DSBs that occur in vivo. To assess the impact of nucleosomes on the frequency and pattern of BER-dependent DSB formation, we incubated nucleosomes containing oxidative damages in opposing DNA strands with selected DNA glycosylases and human apurinic/apyrimidinic endonuclease 1. Overall, nucleosomes substantially suppressed DSB formation. However, the degree of suppression varied as a function of (i) the lesion type and DNA glycosylase tested, (ii) local sequence context and the stagger between opposing strand lesions, (iii) the helical orientation of oxidative lesions relative to the underlying histone octamer, and (iv) the distance between the lesion cluster and the nucleosome edge. In some instances the binding of a BER factor to one nucleosomal lesion appeared to facilitate binding to the opposing strand lesion. DSB formation did not invariably lead to nucleosome dissolution, and in some cases, free DNA ends resulting from DSB formation remained associated with the histone octamer. These observations explain how specific structural and dynamic properties of nucleosomes contribute to the suppression of BER-generated DSBs. These studies also suggest that most BER-generated DSBs will occur in linker DNA and in genomic regions associated with elevated rates of nucleosome turnover or remodeling.

Thermodynamic analysis of self-assembly in coiled-coil biomaterials.

Coiled-coil protein structural motifs have proven amenable to the design of structurally well-defined biomaterials. Mesoscale structural properties can be fairly well predicted based on rules governing the chemical interactions between the helices that define this structural motif. We explore the role of the hydrophobic core residues on the self-assembly of a coiled-coil polymer through a mutational analysis coupled with a salting-out procedure. Because the resultant polymers remain in solution, a thermodynamic approach is applied to characterize the polymer assembly using conventional equations from polymer theory to extract nucleation and elongation parameters. The stabilities and lengths of the polymers are measured using circular dichroism spectropolarimetry, sizing methods including dynamic light scattering and analytical ultracentrifugation, and atomic force microscopy to assess mesoscale morphology. Upon mutating isoleucines at two core positions to serines, we find that polymer stability is decreased while the degree of polymerization is about the same. Differences in results from circular dichroism and dynamic light scattering experiments suggest the presence of a stable intermediate state, and a scheme is proposed for how this intermediate might relate to the monomer and polymer states.