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The most highly conserved nucleotides in D5, an essential active site component of group II introns, consist of an AGC triad, of which the G is invariant. To understand how this G participates in catalysis, the mechanistic contribution of its functional groups was examined. We observed that the exocyclic amine of G participates in ground state interactions that stabilize D5 binding from the minor groove. In contrast, each major groove heteroatom of the critical G (specifically N7 or O6) is essential for chemistry. Thus, major groove atoms in an RNA helix can participate in catalysis, despite their presumed inaccessibility. N7 or O6 of the critical G could engage in critical tertiary interactions with the rest of the intron or they could, together with phosphate oxygens, serve as a binding site for catalytic metal ions.

The RecA protein of Escherichia coli is important for genetic recombination in vivo and can promote synapsis and strand exchange in vitro. The DNA pairing and strand exchange reactions have been well characterized in reactions with circular single strands and linear duplexes, but little is known about these two processes using substrates more characteristic of those likely to exist in the cell. Single-stranded linear DNAs were prepared by separating strands of duplex molecules or by cleaving single-stranded circles at a unique restriction site created by annealing a short defined oligonucleotide to the circle. Analysis by gel electrophoresis and electron microscopy revealed that, in the presence of RecA and single-stranded binding proteins, a free 3' homologous end is essential for stable joint molecule formation between linear single-stranded and circular duplex DNA. (2) have shown that the formation of joint molecules occurs in such a fashion that only the 5' end of the viral (+)-strand is displaced from the duplex DNA (Fig. 1B). Using the same substrates, Cox and Lehman (3) confirmed this polarity by restriction endonuclease analysis of RecApromoted branch migration. West et al. (4) concluded that the polarity was the same using linearized duplex DNA and homologous circular ss DNA that carried a short hybridized fragment. These data indicate that, in reactions involving linear duplex and ss circular DNA substrates, the polarity of RecA-catalyzed strand exchange in vitro is 5'-to-3' relative to the ss DNA.While the pairing of circular ss DNA with linear duplex DNA is a rapid and efficient reaction whose substrates and products are well characterized, it is not clear what relationship exists between these substrates and those found in vivo. It would be of interest to examine the formation of stablejoint molecules in reactions involving a ss DNA substrate possessing a free end because such a substrate is likely to be more representative of recombinogenic DNA existing in the cell than the ss circular DNAs used in previous in vitro studies (2)(3)(4)(5)(6). In this study, the formation ofjoint molecules between linear ss DNA and circular duplex DNA substrates has been analyzed. These studies reveal that homology at the 3' end of the linear ss DNA is essential for stable RecAcatalyzed joint molecule formation. This observation is consistent with some existing biochemical data (4, 7), which indicate an important role for free homologous 3' ends in recombination. However, it apparently contradicts predictions based on the polarity of strand exchange involving circular ss and linear duplex DNA substrates (2-4): if RecAcatalyzed strand exchange proceeds 5'-to-3' relative to the ss DNA, then linear ss DNAs in which homology to the duplex circle is restricted to the 3' end would form joint molecules that dissociate with time, while those in which homology is present at the 5' end would grow and become more stable.Two possible explanations to resolve this apparent paradox and their relevance to our understanding o...

Using an in vitro system in which a 5' splice site (5'SS) RNA oligo (A_AG $ GUAAGUAdT) is capable of inducing formation of U2/U4/U5/U6 snRNP complex we show that this oligo specifically binds to U4/U5/U6 snRNP and cross-links to U6 snRNA in the absence of U2 snRNP. Moreover, 5'SS RNA oligo bound to U4/U5/U6 snRNP is chased to U2/U4/U5/U6 snRNP complex upon addition of U2 snRNP. Recognition of the 5'SS by U4/U5/U6 snRNP correlates with the 5'SS consensus sequence. Unlike the interaction with U1 snRNP, this recognition depends largely on interactions other than RNA-RNA base pairing. Finally, the region of U6 snRNA required for this interaction with U4/U5/U6 snRNP is positioned upstream of stem I in the U4-U6 structure. We propose that the 5'SS-U4/US/U6 snRNP complex is an intermediate in spliceosome assembly and that recognition of the 5'SS by U4/U5/U6 snRNP occurs after the 5'SS-U1 snRNA base pairing is disrupted but before the U4-U6 snRNA structure is destabilized.[Key Words: 5' splice site recognition; spliceosome assembly~ snRNPs] Received May 13, 1994~ revised version accepted July 1, 1994.Removal of introns from nuclear precursor messenger RNA {pre-mRNA} is mediated by a large multicomponent complex called the spliceosome which is composed of U1, U2, U4, U5, and U6 small nuclear ribonucleoprotein particles (snRNPs} and numerous proteins (for review, see Green 1991; Guthrie 1991; Moore et al. 1993).In mammals, spliceosome assembly depends on a consensus sequence at the 5' splice site (5'SS}, a branch site with an adjacent polypyrimidine tract, and a 3'SS consensus sequence. Initially, U 1 snRNP binds via base pairing with the 5'SS and commits the pre-mRNA to the splicing pathway. Subsequently, U2 snRNP binds at the branch site to form splicing complex A, which is converted into splicing complex B upon association of U4/ U5/U6 triple snRNP. Within U4/U5/U6 snRNP, U4, and U6 snRNAs are extensively base paired. Prior to {or concomitant with} the first step of splicing, this U4-U6 base pairing is disrupted and U4 snRNP is released. Because the pairing interaction between U6 and U4 snRNAs has been proposed to negatively regulate U6 and is mutually exclusive with the pairing interaction between U6 and U2 snRNAs, this structural rearrangement could represent the catalytic activation of the spliceosome.

The RAD52 gene of Saccharomyces cerevisiae has previously been shown to be involved in both recombination and DNA repair. Here we report on the cloning of this gene. A plasmid containing a 5.9 kb yeast DNA fragment inserted into the BamH1 site of the YEp13 vector has been isolated and shown to complement the X-ray sensitive phenotype of the rad52-1 mutation. The rad52-1 cells containing the plasmid form larger colonies than similar cells having lost the plasmid. This plasmid has been shown not to complement either the U.V. sensitivity or the recombination defect of the E. coli recA mutation. From the insert various fragments have been subcloned into the YRp7 and YIp5 vectors. Integration events of two of the subclones have been genetically mapped to the chromosomal location of RAD52, indicating that the structural gene has been cloned. A 1.97 kb BamH1 fragment subcloned into YRp7 in one orientation complements the rad52-1 mutation, while the same fragment in the opposite orientation fails to complement. Various other subclones indicate that a BglII site, within the BamH1 fragment, is in the RAD52 gene. This BglII site has been deleted by Sl-nuclease digestion and the resulting deletion inactivates the RAD52 gene. BAL31 deletions from one end of a 1.9 kb Sal1-BamH1 fragment have been isolated; up to 0.9 kb can be deleted without loss of RAD52 activity, indicating that the RAD52 gene is approximately 1 kb or less in length.

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and bbk8@columbia.edu B.B.Konforti and Q.Liu contributed equally to this work Group II introns are ribozymes with a complex tertiary architecture that is of great interest as a model for RNA folding. Domain 5 (D5) is a highly conserved region of the intron that is considered one of the most critical structures in the catalytic core. Despite its central importance, the means by which D5 interacts with other core elements is unclear. To obtain a map of potential interaction sites, dimethyl sulfate was used to footprint regions of the intron that are involved in D5 binding. These studies were complemented by measurements of D5 binding to a series of truncated intron derivatives. In this way, the minimal region of the intron required for strong D5 association was defined and the sites most likely to represent thermodynamically significant positions of tertiary contact were identified. These studies show that ground-state D5 binding is mediated by tertiary contacts to specific regions of D1, including a tetraloop receptor and an adjacent three-way junction. In contrast, D2 and D3 are not found to stabilize D5 association. These data highlight the significance of D1-D5 interactions and will facilitate the identification of specific tertiary contacts between them.