Unified two-metal mechanism of RNA synthesis and degradation by RNA polymerase

In DNA-dependent RNA polymerases, reactions of RNA synthesis and degradation are performed by the same active center (in contrast to DNA polymerases in which they are separate). We propose a unified catalytic mechanism for multisubunit RNA polymerases based on the analysis of its 3'-5' exonuclease reaction in the context of crystal structure. The active center involves a symmetrical pair of Mg(2+) ions that switch roles in synthesis and degradation. One ion is retained permanently and the other is recruited ad hoc for each act of catalysis. The weakly bound Mg(2+) is stabilized in the active center in different modes depending on the type of reaction: during synthesis by the beta,gamma-phosphates of the incoming substrate; and during hydrolysis by the phosphates of a non-base-paired nucleoside triphosphate. The latter mode defines a transient, non-specific nucleoside triphosphate-binding site adjacent to the active center, which may serve as a gateway for polymerization of substrates.     

Mismatch repair in human nuclear extracts. Time courses and ATP requirements for kinetically distinguishable steps leading to tightly controlled 5' to 3' and aphidicolin-sensitive 3' to 5' mispair-provoked excision

Mismatch repair (MMR) systems enhance genomic stability by correcting DNA replication errors. The events in mammalian MMR pathways remain poorly understood. Using HeLa cell nuclear extracts, we analyzed correction of mispairs in circular DNA substrates with single defined nicks and measured excision in the absence of exogenous dNTPs by annealing specific oligonucleotide probes. In reactions initiated by concomitant temperature shift and addition of ATP or Mg(2+) to otherwise complete mixtures on ice, ATP-initiated excision and final error correction lagged behind Mg(2+)-initiated reactions, suggesting a very early requirement for ATP but not its hydrolysis. Subsequent stable commitment (resistance to added excess competitor substrate) began within 30 s, required hydrolyzable ATP, and plateaued after 60-70 s. This may reflect formation of hydrolysis-dependent translocating and/or pre-excision complexes. Excision along shorter nick-mispair paths began 15 s later than commitment. Both 3' to 5' and 5' to 3' excision gaps appeared at rates of approximately 0.0055 of final yields per second, respectively, 30 or 2.5 times the nonspecific excision rates. The lag between 3' to 5' excision gaps at two different positions yielded an excision progress rate of 5.2 nucleotides/s. In both substrates, corrected products appeared at fractional rates of 0.0027 of final yield per second. Aphidicolin, known to inhibit both the DNA synthesis and 3' to 5' exonuclease activities of polymerases delta and epsilon, reduced appearance of 3' to 5' excision tracts roughly 4-fold at 90 microm but had no effect on 5' to 3' excision.     

Duality of polynucleotide substrates for Phi29 DNA polymerase: 3'-->5' RNase activity of the enzyme

Phi29 DNA polymerase is a small DNA-dependent DNA polymerase that belongs to eukaryotic B-type DNA polymerases. Despite the small size, the polymerase is a multifunctional proofreading-proficient enzyme. It catalyzes two synthetic reactions (polymerization and deoxynucleotidylation of Phi29 terminal protein) and possesses two degradative activities (pyrophosphorolytic and 3'-->5' DNA exonucleolytic activities). Here we report that Phi29 DNA polymerase exonucleolyticaly degrades ssRNA. The RNase activity acts in a 3' to 5' polarity. Alanine replacements in conserved exonucleolytic site (D12A/D66A) inactivated RNase activity of the enzyme, suggesting that a single active site is responsible for cleavage of both substrates: DNA and RNA. However, the efficiency of RNA hydrolysis is approximately 10-fold lower than for DNA. Phi29 DNA polymerase is widely used in rolling circle amplification (RCA) experiments. We demonstrate that exoribonuclease activity of the enzyme can be used for the target RNA conversion into a primer for RCA, thus expanding application potential of this multifunctional enzyme and opening new opportunities for RNA detection.     

End labeling procedures

There are two ways to label a DNA molecular; by the ends or all along the molecule. End labeling can be performed at the 3′- or 5′-end. Labeling at the 3′ end is performed by filling 3′-end recessed ends with a mixture or labeled and unlabeled dNTPs using Klenow or T4 DNA polymerases. Both reactions are template dependent. Terminal deoxynucleotide transferase incorporates dNTPs at the 3′ end of any kind of DNA molecule or RNA. Labels incorporated at the 3′-end of the DNA molecule prevent any further extension or ligation to any other molecule, but this can be overcome by labeling the 5′-end of the desired DNA molecule. 5′-end labeling is performed by enzymatic methods (T4 polynucleotide kinase exchange and forward reactions), by chemical modification of sensitized oligonucleotides with phosphoroamidite, or by combined methods. Probe cleanup is recommended when high background problems occur, but caution should be taken not to damage the attached probe with harsh chemicals or by light exposure.     

Recognition of sugar moieties on substrate analogues by DNA polymerase alpha 2-primase from developing cherry salmon (Oncorhynchus masou) testes

Several dCTP or dATP analogues, bearing an azido or amino group on 2'- or 3'-position of its sugar moiety, were examined for their inhibitory effects on DNA polymerase alpha 2-primase from developing cherry salmon (Oncorhynchus masou) testes, and the recognition of sugar moieties of the analogues by primase and related nucleic acid polymerases were compared. Among the dCTP analogues tested, 2'-azido-2',3'-dideoxy CTP inhibited primase strongly and RNA polymerases I and II to lesser extent. Although, the Ki value for primase was larger than those of RNA polymerases, the Ki/Km value for primase was smaller. In contrast, 3'-amino-2',3'-dideoxy CTP selectively inhibited DNA polymerase beta. In dATP analogue series, 3'-amino-3'-deoxy ATP inhibited RNA polymerases I and II very strongly to the same extent as 3'-deoxy ATP. This analogues was a more selective inhibitor for RNA polymerases I and II than 3'-dATP itself.     

A single tyrosine prevents insertion of ribonucleotides in the eukaryotic-type phi29 DNA polymerase.

Three conserved motifs (named A, B and C) have been proposed to form the polymerization active site in all classes of DNA-dependent polymerases. In eukaryotic-type (alpha-like) DNA polymerases, motif A is characterized by the consensus "Dx2SLYP". Mutants in phi29 DNA polymerase residue Tyr254 of this conserved motif had been previously shown to be affected in dNTP binding. Here, we show that a single substitution of Tyr254 into a valine residue enables the enzyme to incorporate ribonucleotide substrates, without affecting its wild-type affinity for dNTPs. Whereas the wild-type enzyme preferred dNTPs more than two million-fold over rNTPs, the mutation of Tyr254 into valine reduced the discrimination for rNTPs up to 1000-fold. In addition to this discrimination mechanism, based on sugar selection, phi29 DNA polymerase is very inefficient when extending an RNA primer terminus, allowing its exonucleolytic degradation. These results indicate that the Tyr254 of phi29 DNA polymerase is responsible for the discrimination against the 2'-OH group of an incoming ribonucleotide. This is the first time that the invariant tyrosine residue of motif A is involved in ribo- versus deoxyribonucleotide discrimination in an eukaryotic-type DNA polymerase.     

Properties of an unusual DNA primase from an archaeal plasmid

Primases are specialized DNA-dependent RNA polymerases that synthesize a short oligoribonucleotide complementary to single-stranded template DNA. In the context of cellular DNA replication, primases are indispensable since DNA polymerases are not able to start DNA polymerization de novo.

The primase activity of the replication protein from the archaeal plasmid pRN1 synthesizes a rather unusual mixed primer consisting of a single ribonucleotide at the 5′ end followed by seven deoxynucleotides. Ribonucleotides and deoxynucleotides are strictly required at the respective positions within the primer. Furthermore, in contrast to other archaeo-eukaryotic primases, the primase activity is highly sequence-specific and requires the trinucleotide motif GTG in the template. Primer synthesis starts outside of the recognition motif, immediately 5′ to the recognition motif. The fidelity of the primase synthesis is high, as non-complementary bases are not incorporated into the primer.

     

DNA strand transfer catalyzed by the 5'-3' exonuclease domain of Escherichia coli DNA polymerase I.

A protein which promotes DNA strand transfer between linear double-stranded M13mp19 DNA and single-stranded viral M13mp19 DNA has been isolated from recA- E.coli. The protein is DNA polymerase I. Strand transfer activity residues in the small fragment encoding the 5'-3' exonuclease and can be detected using a recombinant protein comprising the first 324 amino acids encoded by polA. Either the recombinant 5'-3' exonuclease or intact DNA polymerase I can catalyze joint molecule formation, in reactions requiring only Mg2+ and homologous DNA substrates. Both kinds of reactions are unaffected by added ATP. Electron microscopy shows that the joint molecules formed in these reactions bear displaced single strands and therefore this reaction is not simply promoted by annealing of exonuclease-gapped molecules. The pairing reaction is also polar and displaces the 5'-end of the non-complementary strand, extending the heteroduplex joint in a 5'-3' direction relative to the displaced strand. Thus strand transfer occurs with the same polarity as nick translation. These results show that E.coli, like many eukaryotes, possesses a protein which can promote ATP-independent strand-transfer reactions and raises questions concerning the possible biological role of this function.     

The mutational specificity of the Dbh lesion bypass polymerase and its implications

The Dbh polymerase of Sulfolobus solfataricus is a member of the recently described family of low fidelity DNA polymerases involved in bypass of DNA lesions. To investigate the enzymatic properties of Dbh, we characterized the errors made by this polymerase in vitro. Not only is Dbh much less accurate than the "classical" polymerases, but it showed a remarkable tendency to skip over a template pyrimidine positioned immediately 3' to a G residue, generating a single-base deletion. Single-turnover kinetic measurements suggest possible mechanisms. First, Dbh shows a bias in favor of dCTP, such that the rate of incorporation of dCTP opposite a template G is about 10-fold faster than for the other three dNTPs opposite their complementary partners. On a DNA substrate corresponding to a frameshift hotspot, the rate of frameshift insertion of dCTP opposite a template G that is one residue 5' to the expected templating position is approximately equal to the rate of the non-frameshifted C-dGTP insertion. We suspect that the unusual mutational specificity of Dbh (which is shared with other polymerases from the DinB branch of the bypass polymerase family) may be related to the type of DNA lesion(s) that it serves to bypass in vivo.