Crystal structure of an Okazaki fragment at 2-A resolution.

Abstract

Ini DNA replication, Okazaki fragments are formed as double-stranded intermediates during synthesis of the lagging strand. They are composed of the growing DNA strand primed by RNA and the template strand. The DNA oligonucleotide d(GGGTATACGC) and the chimeric RNA-DNA oligonucleotide r(GCG)d(TATACCC) were combined to form a synthetic Okazaki fragment and its three-dimensional structure was determined by x-ray crystallography. The fragment adopts an overall A-type conformation with 11 residues per turn. Although the base-pair geometry, particularly in the central TATA part, is distorted, there is no evidence for a transition from the A-to the B-type conformation at the junction between RNA-DNA hybrid and DNA duplex. The RNA trimer may, therefore, lock the complete fragment in an A-type conformation.DNA replication is a semiconservative process and, in both prokaryotes and eukaryotes, bidirectional growth of both strands starting from a single origin is the most common mechanism (1-3). At the replication fork one of the new DNA strands, the leading strand, grows continuously in the 5'-to-3' direction. Only one primer at the origin of replication is required by the DNA polymerase to catalyze the addition of nucleotides to the 3'-hydroxyl end ot this strand. However, the complementary strand must be, replicated in a discontinuous way (4, 5), as DNA polymerases cannot initiate new chains but require a 3'-OH terminus of a primer oligo-or polynucleotide (6). This and[ the finding that many RNA polymerases are capable of initiating new chains de novo (7) led to the discovery that RNA-is used for the primers in the synthesis of the lagging strand (8-10). The

A crystallographic map of the transition from B-DNA to A-DNA

“…Thus, the A-type nucleotides at the 5Ј-end of each strand are paired with B-type nucleotides at the 3Ј-end of the complementary strand. This mixture of A-and B-DNA base pairing is similar to the A-RNA͞B-DNA chimera seen by fiber diffraction (24), but differs from the A-RNA structures of DNA͞RNA hybrids (25,26). The composite conformation seen here is analogous to the junction between B-and A-DNA first proposed by Arnott and colleagues (27) 2 , both of which had extended helical rises compared with both A-and B-DNAs (6).…”

Macromolecular Cryocrystallography

“…The hydrocarbon oil protects the crystal during transfer and on solidification acts as a glue between the crystal and the glass fibre. This method has been used for the structure determination of numerous DNA oligomers (Eisenstein, Hope, Haran, Frolow, Shakked & Rabinovich, 1988;Joshua-Tor, Rabinovich, Hope, Frolow, Appella & Sussman, 1988;Shakked, Guerstein-Guzikevich, Eisenstein, Frolow & Rabinovich, 1989;Eisenstein, Frolow, Shakked & Rabinovich, 1990;Narayana, Ginell, Russu & Berman;Quintana, Lipanov & Dickerson, 1991;Joshua-Tor, Frolow, Appella, Hope, Rabinovich & Sussmann, 1992;Ginell, Vojtechovsky, Gaffney, Jones & Berman, 1994;Bancroft, Williams, Rich & Egli, 1994) and a large number of proteins (Teeter & Hope, 1986;Wagner, Werber, Beck, Hartman, Frolow & Sussmann, 1989;Freymann, Down, Carrington, Roditi, Turner & Wiley, 1990;Livnah & Sussman, 1990;Earnest, Fauman, Craik & Stroud, 1991;Wilke, Higaki, Craik & Fletterick, 1991;Watt, Tulinsky, Swenson & Watenpaugh, 1991;Harel, Su, Frolow, Ashani, Silman & Sussman, 1991;Harel, Su, Frolow, Silman & Sussmann, 1991;Chen& Herzberg, 1992;Day, Hsu, Joshua-Tor, Park, Zhou, Adams & Rees, 1992;Egli, Usman, Zhang & Rich, 1992;Georgiadis, Komiya, Chakrabarti, Woo, Kornuc & Rees, 1992;Rini, Schulze-Gahmen & Wilson, 1992;Kim, Kwon, Myers & Rees, 1993;Lima, Wang & Mondragon, 1994;Knegtel, Strokopytov, Penninga, Faber, Rozeboom, Kalk, Dijkhuizen & Dijkstraa, 1995;…”

RNA – Synthesis, Purification and Crystallization

“…RNA sequences up to about 80 nucleotides can now be synthesized and purified (Gasparutto et al,great ease of obtaining heavy-atom derivatives using halogenated phosphoramidites was demonstrated . Reports on successful crystallizations (Baeyens, Jancarik & Holbrook, 1994;Doudna et al, 1993;Kim et al, 1994;Pley, Lindes, DeLuca-Flaherty & McKay, 1993; and structure determinations of RNA (Baeyens, De Bondt & Holbrook, 1995;Betzel et al, 1994;Cruse et al, 1994;Dock-Bregeon et al, 1988Holbrook et al, 1991;Leonard et al, 1994;Pley et al, 1994;Portmann, Usman & Egli, 1995;Schindelin et al, 1995;Wahl, Ban, Sekharudu, Ramakrishnan & Sundaralingam, 1996;Wahl, Rao & Sundaralingam, 1996) and DNA-RNA chimeras (Ban, Ramakrishnan & Sundaralingam, 1994a,b;Chen, Ramakrishnan & Sundaralingam, 1995;Egli, Usman & Rich, 1993;Egli, Usman, Zhang & Rich, 1992;Wang et al, 1982) are now appearing in increased numbers in the literature, illustrating the importance of the field. New, reliable protocols for the production and crystallization of RNA's are therefore still in need, to support structure-function investigations by NMR and X-ray crystallography.…”