RNA-DNA hybrids containing damaged DNA are substrates for RNase H.

During the replication of the lagging strand, RNA-DNA hybrids are formed and the RNA is subsequently degraded by the action of RNase H. Little is known about the effects of damaged DNA on lagging strand replication and subsequent RNA removal. The rates and sites of digestion by E. coli RNase H of RNA-DNA hybrids containing either a thymine glycol or urea site in the DNA strand have been examined. The cleavage patterns for duplexes containing thymine glycol or urea differ from that of a fully complementary duplex. There is one major product of the digestion of the fully complementary hybrid, but three products are formed in the reactions with the hybrids containing damaged DNAs. Cleavage is partially redirected to the position adjacent to the damaged sites. The overall rate of cleavage of these hybrids containing damaged DNA is comparable to that of the fully complementary duplex. These results indicate that the cleavage of RNA-DNA hybrids by RNase H is less selective when a damaged site is present in the DNA strand.

Distance measurements in nucleic acids using windowless dipolar recoupling solid state NMR.

A windowless, homonuclear dipolar recoupling pulse sequence (DRAWS) is described and a theoretical basis for describing its recoupling performance is developed using numerical techniques. It is demonstrated that DRAWS recouples weak dipolar interactions over a broad range of experimental and molecular conditions. We discuss two spectroscopic control experiments, which help to take into account effects due to insufficient proton decoupling, relaxation, and static dipolar couplings to nearby 13C spins at natural abundance. Finally DRAWS is used in combination with selective 13C labeling to measure 13C-13C distances in five doubly labeled DNA dodecamers, [d(CGCGAAT*T*CGCG)]2, which contain the binding site for the restriction enzyme EcoRI. The longest distance reported is 4.8 A. In most cases the distances agree well with those derived from X-ray crystallographic data, although small changes in hydration level can result in relatively large changes in internuclear distances.