Structure of a pseudo-16-mer DNA with stacked guanines and two G-A mispairs complexed with the N-terminal fragment of Moloney murine leukemia virus reverse transcriptase

The X-ray crystal structure at 2.0 Angstrom resolution of a DNA molecule co mplexed with the N-terminal fragment of Moloney murine leukemia virus rever se transcriptase (MMLV RT) has been determined. This method allows the stud y of nucleic acids in a unique and largely unfettered environment without t he complicated lattice interactions typically observed in DNA-only crystal structures. Molecular-replacement phasing using only the protein provided r eadily interpretable electron density with no model bias for the DNA. The a symmetric unit of the structure consists of the protein molecule bound to t he blunt end of a DNA 6/10-mer, which is composed of a six-base strand (5'- GTCGTC-3') and a ten-base strand (3'-CAGCAGGGCA-5'), resulting in a six-bas e-pair duplex with a four-base single-stranded overhang. In the crystal str ucture, the bases of the overhang reciprocally pair to yield a doubly nicke d pseudo-hexadecamer primarily B-form DNA molecule. The pairing between the single strands gives two standard (G-C) Watson-Crick pairs and two G(anti) -A(anti) mispairs. The mispairs reside in a G-C-rich environment and the th ree consecutive guanines on the 10-mer impart interesting structural featur es to the pseudo-hexadecamer, such as the preference for a guanine stack, s tretching the C-G base pairs flanking the mispair to the point of loss of i ntra-base-pair hydrogen bonding. The DNA was designed for the purpose of co mparison with a previous structure, which was determined in the same crysta l lattice. In all of the authors' previous fragment-DNA complexes, the nucl eotide at the blunt-ended 3'-hydroxyl was a purine. Consistent with the pro posed mechanistic role of interactions with the 3'-hydroxyl in processive D NA synthesis by RT, it was found that a pyrimidine at this position in the DNA makes indentical interactions with the strictly conserved Gly191 and th e main chain of Leu115 of MMLV RT.