Eukaryotic Mismatch Repair in Relation to DNA Replication

Three processes act in series to accurately replicate the eukaryotic nuclear genome. The major replicative DNA polymerases strongly prevent mismatch formation, occasional mismatches that do form are proofread during replication, and rare mismatches that escape proofreading are corrected by mismatch repair (MMR). This review focuses on MMR in light of increasing knowledge about nuclear DNA replication enzymology and the rate and specificity with which mismatches are generated during leading- and lagging-strand replication. We consider differences in MMR efficiency in relation to mismatch recognition, signaling to direct MMR to the nascent strand, mismatch removal, and the timing of MMR. These studies are refining our understanding of relationships between generating and repairing replication errors to achieve accurate replication of both DNA strands of the nuclear genome

Downstream DNA Sequence Effects on Transcription Elongation: ALLOSTERIC BINDING OF NUCLEOSIDE TRIPHOSPHATES FACILITATES TRANSLOCATION VIA A RATCHET MOTION

The ability of RNA polymerase (RNAP) to adopt multiple conformations is central to transcriptional regulation. In previous work, we demonstrated that RNAP can exist in an unactivated state that catalyzes synthesis slowly and an activated state that catalyzes synthesis rapidly, with the transition from the unactivated to the activated state being induced by the templated NTP binding to an allosteric site on the RNAP. In this work, we investigate the effects of downstream DNA sequences on the kinetics of single nucleotide incorporation. We demonstrate that changing the identity of the DNA base 1 bp downstream (+2) from the site of incorporation (+1) can regulate the catalytic activity of RNAP. Combining these data with sequence and structural analyses and molecular modeling, we identify the streptolydigin-binding region (Escherichia coli beta residues 543-546), which lies across from the downstream DNA, as the putative allosteric NTP binding site. We present a structural model in which the NTP binds to the streptolydigin loop and upon pairing with the +1 DNA base in the unactivated state or the +2 DNA base in the activated state facilitates translocation via a ratchet motion. This model provides an alternative mechanism for pausing as well as a structural explanation not only for our kinetic data but also for data from elongation studies on yeast RNAP II

Single molecule studies of DNA mismatch repair

DNA mismatch repair involves is a widely conserved set of proteins that is essential to limit genetic drift in all organisms. The same system of proteins plays key roles in many cancer related cellular transactions in humans. Although the basic process has been reconstituted in vitro using purified components, many fundamental aspects of DNA mismatch repair remain hidden due in part to the complexity and transient nature of the interactions between the mismatch repair proteins and DNA substrates. Single molecule methods offer the capability to uncover these transient but complex interactions and allow novel insights into mechanisms that underlie DNA mismatch repair. In this review, we discuss applications of single molecule methodology including electron microscopy, atomic force microscopy, particle tracking, FRET, and optical trapping to studies of DNA mismatch repair. These studies have led to formulation of mechanistic models of how proteins identify single base mismatches in the vast background of matched DNA and signal for their repair

Forks, pincers, and triggers: the tools for nucleotide incorporation and translocation in multi-subunit RNA polymerases

The central role of RNA polymerase (RNAP) is to catalyze the processive synthesis of a growing RNA transcript. Recent structural and biophysical data have lead to a deeper understanding of the nucleotide addition cycle and insight into the structure-function relationships that govern transcription elongation. In this review, we discuss kinetic data on nucleotide incorporation in the context of crystal structures, which show RNAP in multiple conformations. We present a facilitated Brownian ratchet model of nucleotide incorporation, in which templated NTP binding to a non-catalytic site in the main channel promotes the conformational changes that lead to opening of the catalytic site and translocation

Determination of protein–DNA binding constants and specificities from statistical analyses of single molecules: MutS–DNA interactions

Atomic force microscopy (AFM) is a powerful technique for examining the conformations of protein–DNA complexes and determining the stoichiometries and affinities of protein–protein complexes. We extend the capabilities of AFM to the determination of protein–DNA binding constants and specificities. The distribution of positions of the protein on the DNA fragments provides a direct measure of specificity and requires no knowledge of the absolute binding constants. The fractional occupancies of the protein at a given position in conjunction with the protein and DNA concentrations permit the determination of the absolute binding constants. We present the theoretical basis for this analysis and demonstrate its utility by characterizing the interaction of MutS with DNA fragments containing either no mismatch or a single mismatch. We show that MutS has significantly higher specificities for mismatches than was previously suggested from bulk studies and that the apparent low specificities are the result of high affinity binding to DNA ends. These results resolve the puzzle of the apparent low binding specificity of MutS with the expected high repair specificities. In conclusion, from a single set of AFM experiments, it is possible to determine the binding affinity, specificity and stoichiometry, as well as the conformational properties of the protein–DNA complexes

Transcript Cleavage by Thermus thermophilus RNA Polymerase: EFFECTS OF GreA AND ANTI-GreA FACTORS

All known multisubunit RNA polymerases possess the ability to endonucleolytically degrade the nascent RNA transcript. To gain further insight into the conformational changes that govern transcript cleavage, we have examined the effects of certain anions on the intrinsic transcript cleavage activity of Thermus thermophilus RNA polymerase. Our results indicate that the conformational transitions involved in transcript cleavage, and therefore backtracking, are anion-dependent. In addition to characterizing the intrinsic cleavage activity of T. thermophilus RNA polymerase, we have identified, cloned, and expressed a homolog of the prokaryotic transcript cleavage factor GreA from the extreme thermophiles, T. thermophilus and Thermus aquaticus. The thermostable GreA factors contact the 3'-end of RNA, stimulate the intrinsic cleavage activity of T. thermophilus RNA polymerase, and increase the k(app) of the cleavage reaction 25-fold. In addition, we have identified a novel transcription factor in T. thermophilus and T. aquaticus that shares a high degree of sequence similarity with GreA, but has several residues that are not conserved with the N-terminal "basic patch" region of GreA. This protein, Gfh1, functions as an anti-GreA factor in vitro by reducing intrinsic cleavage and competing with GreA for a binding site on the polymerase

Human Factor IX Binds to Specific Sites on the Collagenous Domain of Collagen IV

The primary region of factor IX that mediates binding to bovine aortic endothelial cells resides in residues 3-11 of the N-terminal region known as the Gla domain. Recently, it was proposed that the observed binding to endothelial cells is actually a measure of the interaction between factor IX and collagen IV (Cheung, W. F., van den Born, J., Kuhn, K., Kjellen, L., Hudson, B. G., and Stafford, D. W. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 11068-11073). To confirm that factor IX binds to collagen IV and to examine the specificity of this interaction, we used scanning force microscopy to examine factor IX binding to collagen IV. We imaged collagen IV in the presence and the absence of factor IX and observed specific interactions between factor IX and collagen IV. Our results demonstrate that factor IX binds to collagen IV at specific sites in the collagenous domain approximately 98 and approximately 50 nm from the C-terminal pepsin-cleaved end

Visualizing the Path of DNA through Proteins Using DREEM Imaging

Many cellular functions require the assembly of multiprotein-DNA complexes. A growing area of structural biology aims to characterize these dynamic structures by combining atomic-resolution crystal structures with lower-resolution data from techniques that provide distributions of species, such as small-angle X-ray scattering, electron microscopy, and atomic force microscopy (AFM). A significant limitation in these combinatorial methods is localization of the DNA within the multiprotein complex. Here, we combine AFM with an electrostatic force microscopy (EFM) method to develop an exquisitely sensitive dual-resonance-frequency-enhanced EFM (DREEM) capable of resolving DNA within protein-DNA complexes. Imaging of nucleosomes and DNA mismatch repair complexes demonstrates that DREEM can reveal both the path of the DNA wrapping around histones and the path of DNA as it passes through both single proteins and multiprotein complexes. Finally, DREEM imaging requires only minor modifications of many existing commercial AFMs, making the technique readily available

Interconvertible Lac Repressor–DNA Loops Revealed by Single-Molecule Experiments

At many promoters, transcription is regulated by simultaneous binding of a protein to multiple sites on DNA, but the structures and dynamics of such transcription factor-mediated DNA loops are poorly understood. We directly examined in vitro loop formation mediated by Escherichia coli lactose repressor using single-molecule structural and kinetics methods. Small (∼150 bp) loops form quickly and stably, even with out-of-phase operator spacings. Unexpectedly, repeated spontaneous transitions between two distinct loop structures were observed in individual protein–DNA complexes. The results imply a dynamic equilibrium between a novel loop structure with the repressor in its crystallographic “V” conformation and a second structure with a more extended linear repressor conformation that substantially lessens the DNA bending strain. The ability to switch between different loop structures may help to explain how robust transcription regulation is maintained even though the mechanical work required to form a loop may change substantially with metabolic conditions

Single-Molecule FRET TACKLE Reveals Highly Dynamic Mismatched DNA−MutS Complexes

The first step in DNA mismatch repair (MMR) is the recognition of DNA mismatches or nucleotide insertions/deletions (IDLs) by MutS and MutS homologs. To investigate the conformational properties of MutS-mismatch complexes, we used single-molecule fluorescence resonance energy transfer (smFRET) to examine the dynamics of MutS-induced DNA bending at a GT-mismatch. The FRET measurements reveal that the MutS-GT-mismatch recognition complex is highly dynamic, undergoing conformational transitions between many states with different degrees of DNA bending. Due to the complexity of the data, we developed an analysis approach, called FRET TACKLE, in which we combine direct analysis of FRET transitions with examination of kinetic lifetimes to identify all of the conformational states and characterize the kinetics of the binding and conformational equilibria. The data reveal that MutS-GT complexes can reside in six different conformations, which have lifetimes that differ by as much as 20-fold and exhibit rates of interconversion that vary by two orders of magnitude. To gain further insight into the dynamic properties of GT-MutS complexes and to bolster the validity of our analysis, we complemented our experimental data with Monte Carlo simulations. Taken together, our results suggest that the dynamics of the MutS-mismatch complex could govern the efficiency of repair of different DNA mismatches. Finally, in addition to revealing these important biological implications of MutS-DNA interactions, this FRET TACKLE method will enable the analysis of the complex dynamics of other biological systems