Multiple DNA-binding sites in Tetrahymena telomerase

Telomerase is a ribonucleoprotein enzyme that maintains chromosome ends through de novo addition of telomeric DNA. The ability of telomerase to interact with its DNA substrate at sites outside its catalytic centre (‘anchor sites’) is important for its unique ability to undergo repeat addition processivity. We have developed a direct and quantitative equilibrium primer-binding assay to measure DNA-binding affinities of regions of the catalytic protein subunit of recombinant Tetrahymena telomerase (TERT). There are specific telomeric DNA-binding sites in at least four regions of TERT (the TEN, RBD, RT and C-terminal domains). Together, these sites contribute to specific and high-affinity DNA binding, with a K_d of ~8 nM. Both the K_m and K_d increased in a stepwise manner as the primer length was reduced; thus recombinant Tetrahymena telomerase, like the endogenous enzyme, contains multiple anchor sites. The N-terminal TEN domain, which has previously been implicated in DNA binding, shows only low affinity binding. However, there appears to be cooperativity between the TEN and RNA-binding domains. Our data suggest that different DNA-binding sites are used by the enzyme during different stages of the addition cycle.     

DNA polymerase III holoenzyme from Thermus thermophilus identification, expression, purification of components, and use to reconstitute a processive replicase

DNA replication in bacteria is performed by a specialized multicomponent replicase, the DNA polymerase III holoenzyme, that consist of three essential components: a polymerase, the beta sliding clamp processivity factor, and the DnaX complex clamp-loader. We report here the assembly of the minimal functional holoenzyme from Thermus thermophilus (Tth), an extreme thermophile. The minimal holoenzyme consists of alpha (pol III catalytic subunit), beta (sliding clamp processivity factor), and the essential DnaX (tau/gamma), delta and delta' components of the DnaX complex. We show with purified recombinant proteins that these five components are required for rapid and processive DNA synthesis on long single-stranded DNA templates. Subunit interactions known to occur in DNA polymerase III holoenzyme from mesophilic bacteria including delta-delta' interaction, deltadelta'-tau/gamma complex formation, and alpha-tau interaction, also occur within the Tth enzyme. As in mesophilic holoenzymes, in the presence of a primed DNA template, these subunits assemble into a stable initiation complex in an ATP-dependent manner. However, in contrast to replicative polymerases from mesophilic bacteria, Tth holoenzyme is efficient only at temperatures above 50 degrees C, both with regard to initiation complex formation and processive DNA synthesis. The minimal Tth DNA polymerase III holoenzyme displays an elongation rate of 350 bp/s at 72 degrees C and a processivity of greater than 8.6 kilobases, the length of the template that is fully replicated after a single association event.     

Delineation of the high-affinity single-stranded telomeric DNA-binding domain of Saccharomyces cerevisiae Cdc13

Cdc13 is an essential protein from Saccharomyces cerevisiae that caps telomeres by protecting the C-rich telomeric DNA strand from degradation and facilitates telomeric DNA replication by telomerase. In vitro, Cdc13 binds TG-rich single-stranded telomeric DNA with high affinity and specificity. A previously identified domain of Cdc13 encompassing amino acids 451–694 (the 451–694 DBD) retains the single-stranded DNA-binding properties of the full-length protein; however, this domain contains a large unfolded region identified in heteronuclear NMR experiments. Trypsin digestion and MALDI mass spectrometry were used to identify the minimal DNA-binding domain (the 497–694 DBD) necessary and sufficient for full DNA-binding activity. This domain was completely folded, and the N-terminal unfolded region removed was shown to be dispensable for function. Using affinity photocrosslinking to site-specifically modified telomeric single-stranded DNA, the 497–694 DBD was shown to contact the entire 11mer required for high-affinity binding. Intriguingly, both domains bound single-stranded telomeric DNA with much greater affinity than the full-length protein. The full-length protein exhibited the same rate of dissociation as both domains, however, indicating that the full-length protein contains a region that inhibits association with single-stranded telomeric DNA.     

A possible mechanism of processive nucleotide and repeat additions by the telomerase

Telomerase is a specialized cellular ribonucleoprotein complex that can synthesize long stretches of a DNA primer by using an intrinsic RNA template sequence. This requires that the telomerase must be able to carry out both nucleotide and repeat additions. Here, based on available structures and experimental data, a model is presented to describe these two addition activities. In the model, the forward movement of the polymerase active site along the template during the processive nucleotide addition is rectified through the incorporation of a matched base, via the Brownian ratchet mechanism. The unpairing of the DNA:RNA hybrid and then repositioning of product 3′-end after each round of repeat synthesis, which are prerequisites for the processive repeat addition, are caused by a force acting on the primer. The force results from the conformational transition of the stem III pseudoknot, which is mechanically induced by the rotation of TERT fingers together with stem IV loop towards the polymerase active site upon a nucleotide binding. Based on the model, the dynamics of processive nucleotide and repeat additions by recombinant Tetrahymena telomerase is studied analytically, which gives good quantitative explanations to the previous experimental results. Moreover, some predicted results are presented. In particular, it is shown that the repeat addition processivity is mainly determined by the difference between the free-energy change required to disrupt the DNA:RNA hybrid and that required to unfold the stem III pseudoknot. A large difference in free energy corresponds to a low repeat addition processivity while a small difference in free energy corresponds to a high repeat addition processivity.     

The Middle Subunit of Replication Protein A Contacts Growing RNA-DNA Primers in Replicating Simian Virus 40 Chromosomes

The eukaryotic single-stranded DNA binding protein replication protein A (RPA) participates in major DNA transactions. RPA also interacts through its middle subunit (Rpa2) with regulators of the cell division cycle and of the response to DNA damage. A specific contact between Rpa2 and nascent simian virus 40 DNA was revealed by in situ UV cross-linking. The dynamic attributes of the cross-linked DNA, namely, its size distribution, RNA primer content, and replication fork polarity, were determined. These data suggest that Rpa2 contacts the early DNA chain intermediates synthesized by DNA polymerase α-primase (RNA-DNA primers) but not more advanced products. Possible signaling functions of Rpa2 are discussed, and current models of eukaryotic lagging-strand DNA synthesis are evaluated in view of our results.     

Modular construction for function of a ribonucleoprotein enzyme: the catalytic domain of Bacillus subtilis RNase P complexed with B. subtilis RNase P protein

The bacterial RNase P holoenzyme catalyzes the formation of the mature 5′-end of tRNAs and is composed of an RNA and a protein subunit. Among the two folding domains of the RNase P RNA, the catalytic domain (C-domain) contains the active site of this ribozyme. We investigated specific binding of the Bacillus subtilis C-domain with the B.subtilis RNase P protein and examined the catalytic activity of this C-domain–P protein complex. The C-domain forms a specific complex with the P protein with a binding constant of ~0.1 µM. The C-domain–P protein complex and the holoenzyme are equally efficient in cleaving single-stranded RNA (~0.9 min^–1 at pH 7.8) and substrates with a hairpin–loop 3′ to the cleavage site (~40 min^–1). The holoenzyme reaction is much more efficient with a pre-tRNA substrate, binding at least 100-fold better and cleaving 10–500 times more efficiently. These results demonstrate that the RNase P holoenzyme is functionally constructed in three parts. The catalytic domain alone contains the active site, but has little specificity and affinity for most substrates. The specificity and affinity for the substrate is generated by either the specificity domain of RNase P RNA binding to a T stem–loop-like hairpin or RNase P protein binding to a single-stranded RNA. This modular construction may be exploited to obtain RNase P-based ribonucleoprotein complexes with altered substrate specificity.