Recent studies indicate that replicative DNA polymerases frequently incorporate ribonucleotides into DNA during replication of the nuclear genome in S. cerevisiae3
and in S. pombe
The major roles of DNA polymerases epsilon and delta at the eukaryotic replication fork are evolutionarily conserved.42
Some consequences of ribonucleotide incorporation become unmasked in yeast strains lacking RNase H2, which initiates efficient repair of newly incorporated ribonucleotides. Such strains exhibit the characteristics of replicative stress, including slow progression through S phase and genome instability.5
The present investigation was motivated by the need to understand the structural implications of the presence of such isolated ribonucleotides in DNA. There have been several reported studies of the structural perturbations resulting from the introduction of isolated ribonucleotides into double stranded DNA.17,19–22
In each case, the crystallographic result was a global conversion of the entire DNA strand from B-form to A-form, even in the cases in which the ribonucleotide was present at the terminal position. Alternatively, in the one previously reported NMR study of a short sequence containing an isolated cytidine nucleoside, the structural perturbation was much more localized.17
In the present study, we have investigated the structure of the extensively characterized Dickerson dodecamer, modified here to contain a single guanosine ribonucleotide at (equivalent) positions 4 and 21.
Even prior to any structural analysis, the highly localized nature of the perturbation introduced by the ribonucleotide is apparent from a comparison of the 1H shift data and 3JHH data with the results reported for the unsubstituted dodecamer (, ). Excluding the modified position and its two nearest neighbors, all of the proton shifts in the sugar moieties are within 0.1 ppm, and in most cases, much smaller. The ribonucleotide substitution increases the value of the A5 3J1'2' by 1.7 Hz, and reduces rG4-A5 3J3‘p scalar coupling by −3.15 Hz, but introduces no other variations greater than 1 Hz.
The results of the NMR study are supported by a series of AMBER calculations, which also indicate the localized nature of the conformational perturbation introduced by the 2'-OH group. The overall conclusions are similar to those reported for studies of DNA containing an isolated arabinonucleoside,24
as well as theoretical analyses,44
all of which show only localized structural perturbations that do not significantly alter the global B-form conformation. The localized nature of the structural perturbation is also consistent with recent structural data for an RNase H2-rcDNA complex.45
The rcDNA present in the complex, with sequence: d(GACAC)r(C)d(TGATTC)•d(GAATCAGGTGTC) is characterized by a stretch of approximately 7 nucleotides at the protein interface, centered around the ribonucleotide, that adopt an A-form conformation. Nevertheless, although the DNA-enzyme interactions are sufficient to induce A-form geometry over a localized 7-nucleotide segment, the DNA residues beyond this region exhibit a minor groove width more characteristic of B-DNA. Hence, even with the more extensive enzyme-induced A-form geometry, there is no global transition to A-form DNA. Of course, it is not unusual for macromolecular interactions to strongly perturb DNA conformation.
The factors that bias crystal structures toward A-form DNA have been well discussed in the literature and include dehydration and cationic salts that reduce electrostatic repulsion of the phosphate groups in the major groove.46–51
However, such factors would also be operative for the unsubstituted Dickerson dodecamer, which was also crystallized in the presence of spermine and MPD; yet this sequence has been observed to adopt a B-form geometry in multiple crystallographic studies.23,52
The present results for the well studied dd-DNA sequence also demonstrate that the inconsistent method-dependent results are not a consequence of differences in nucleotide sequence, as has also been suggested.53
The introduction of the isolated ribonucleotide may increase the probability of global A-form rcDNA conformers, however the NMR parameters for nucleotides further than ± 1 position from the substitution do not reveal any significantly increased A-form bias. One constraint that has received less explicit discussion but appears to be of particular relevance to the present study involves the symmetry preferences at lattice contacts. The repetitive structural regularity of DNA characterized by a consistent global geometry allows for multiple lattice contacts along the length of the molecule. From this perspective, either A-form or B-form rcDNA is preferable to B-form rcDNA with a localized A-form perturbation. If the less symmetric form identified in solution does not crystallize well, then the crystal selection will be between the more regular A- and B-forms (). Due to the significant energy penalty of placing the ribonucleotide in a B-form geometry, any B-form rcDNA that forms is expected to be very short lived. In summary, it is likely that lattice packing constraints that favor conformational regularity, rather than more general effects that bias DNA structures toward A-form geometry, represent the primary basis for the systematic difference between crystallographic and solution-based determinations for the structure of dsDNA containing isolated ribonucleotides.