Mismatched DNA Substrates Research Articles

The Startling Role of Mismatch Repair in Trinucleotide Repeat Expansions.

Trinucleotide repeats are a peculiar class of microsatellites whose expansions are responsible for approximately 30 human neurological or developmental disorders. The molecular mechanisms responsible for these expansions in humans are not totally understood, but experiments in model systems such as yeast, transgenic mice, and human cells have brought evidence that the mismatch repair machinery is involved in generating these expansions. The present review summarizes, in the first part, the role of mismatch repair in detecting and fixing the DNA strand slippage occurring during microsatellite replication. In the second part, key molecular differences between normal microsatellites and those that show a bias toward expansions are extensively presented. The effect of mismatch repair mutants on microsatellite expansions is detailed in model systems, and in vitro experiments on mismatched DNA substrates are described. Finally, a model presenting the possible roles of the mismatch repair machinery in microsatellite expansions is proposed.

Kinetic investigation of the polymerase and exonuclease activities of human DNA polymerase ε holoenzyme

In eukaryotic DNA replication, DNA polymerase ε (Polε) is responsible for leading strand synthesis, whereas DNA polymerases α and δ synthesize the lagging strand. The human Polε (hPolε) holoenzyme is comprised of the catalytic p261 subunit and the noncatalytic p59, p17, and p12 small subunits. So far, the contribution of the noncatalytic subunits to hPolε function is not well understood. Using pre-steady-state kinetic methods, we established a minimal kinetic mechanism for DNA polymerization and editing catalyzed by the hPolε holoenzyme. Compared with the 140-kDa N-terminal catalytic fragment of p261 (p261N), which we kinetically characterized in our earlier studies, the presence of the p261 C-terminal domain (p261C) and the three small subunits increased the DNA binding affinity and the base substitution fidelity. Although the small subunits enhanced correct nucleotide incorporation efficiency, there was a wide range of rate constants when incorporating a correct nucleotide over a single-base mismatch. Surprisingly, the 3'→5' exonuclease activity of the hPolε holoenzyme was significantly slower than that of p261N when editing both matched and mismatched DNA substrates. This suggests that the presence of p261C and the three small subunits regulates the 3'→5' exonuclease activity of the hPolε holoenzyme. Together, the 3'→5' exonuclease activity and the variable mismatch extension activity modulate the overall fidelity of the hPolε holoenzyme by up to 3 orders of magnitude. Thus, the presence of p261C and the three noncatalytic subunits optimizes the dual enzymatic activities of the catalytic p261 subunit and makes the hPolε holoenzyme an efficient and faithful replicative DNA polymerase.

Visualizing Spontaneous DNA Dynamics and its Role in Mismatch Recognition by Damage Recognition Protein Rad4

How DNA-binding proteins search for and identify their target sites buried amid a vast excess of non-target sites remains a central question in biology. This study is aimed at elucidating the mechanism by which DNA damage recognition protein XPC recognizes diverse DNA lesions, and initiates DNA repair. Structural studies of Rad4 (yeast ortholog of XPC) bound to model lesions showed that DNA is unwound and bent at the lesion site and the damaged nucleotides are flipped out. Previous studies using laser T-jump perturbation approaches indicated that Rad4 interrogates potential sites by unwinding DNA on ~100-500 μs and eventually flips out damaged nucleotides on ~5-10 ms. However, these ‘ensemble’ measurements could not capture intrinsic DNA deformability that is potentially critical in the ability of Rad4/XPC to distinguish damaged from undamaged sites. Previous studies were also limited to measurements on short, linear DNA oligomers, while DNA in our cells is typically bent and supercoiled, which is expected to have a profound impact on DNA damage recognition. In this study, I employed an innovative fluorescence lifetime approach with fluorescent probes exquisitely sensitive to DNA twisting/bending fluctuations and mapped the range of conformations accessible to mismatched DNA substrates of varying Rad4-binding specificities. My results reveal a direct connection between intrinsic DNA deformability and Rad4 recognition. High-specificity mismatched DNA, free in solution, sampled a strikingly broad range of conformations from B-DNA-like to highly distorted conformations that resembled those observed with Rad4 bound. Conversely, nonspecific mismatched DNA retained a largely homogeneous, B-DNA-like conformation. Surprisingly, even mismatched-DNA specifically bound to Rad4 remained highly dynamic, a feature that may reflect the versatility of Rad4/XPC to recognize many structurally dissimilar lesions. Using T-jump, I unveiled the rates of intrinsic DNA conformational dynamics in mismatched DNA. Remarkably, these rates remained unaltered in the presence of Rad4. Finally, I examined the effect of DNA bending strain on the intrinsic deformability at the mismatched sites by extending the fluorescence lifetime studies in the context of DNA minicircles. These studies revealed amplified distortions for both matched and mismatched sites in DNA minicircles, with further distortions detected upon Rad4 binding to mismatched sites. Taken together, my results strongly suggest that intrinsic DNA deformability promotes Rad4 damage recognition, perhaps by stalling a diffusing protein and/or facilitating ‘conformational capture’ of pre-distorted damaged sites.

Enhanced spontaneous DNA twisting/bending fluctuations unveiled by fluorescence lifetime distributions promote mismatch recognition by the Rad4 nucleotide excision repair complex.

Rad4/XPC recognizes diverse DNA lesions including ultraviolet-photolesions and carcinogen-DNA adducts, initiating nucleotide excision repair. Studies have suggested that Rad4/XPC senses lesion-induced helix-destabilization to flip out nucleotides from damaged DNA sites. However, characterizing how DNA deformability and/or distortions impact recognition has been challenging. Here, using fluorescence lifetime measurements empowered by a maximum entropy algorithm, we mapped the conformational heterogeneities of artificially destabilized mismatched DNA substrates of varying Rad4-binding specificities. The conformational distributions, as probed by FRET between a cytosine-analog pair exquisitely sensitive to DNA twisting/bending, reveal a direct connection between intrinsic DNA deformability and Rad4 recognition. High-specificity CCC/CCC mismatch, free in solution, sampled a strikingly broad range of conformations from B-DNA-like to highly distorted conformations that resembled those observed with Rad4 bound; the extent of these distortions increased with bound Rad4 and with temperature. Conversely, the non-specific TAT/TAT mismatch had a homogeneous, B-DNA-like conformation. Molecular dynamics simulations also revealed a wide distribution of conformations for CCC/CCC, complementing experimental findings. We propose that intrinsic deformability promotes Rad4 damage recognition, perhaps by stalling a diffusing protein and/or facilitating ‘conformational capture’ of pre-distorted damaged sites. Surprisingly, even mismatched DNA specifically bound to Rad4 remains highly dynamic, a feature that may reflect the versatility of Rad4/XPC to recognize many structurally dissimilar lesions.

Exonuclease processivity of archaeal replicative DNA polymerase in association with PCNA is expedited by mismatches in DNA

Family B DNA polymerases comprise polymerase and 3′ −>5′ exonuclease domains, and detect a mismatch in a newly synthesized strand to remove it in cooperation with Proliferating cell nuclear antigen (PCNA), which encircles the DNA to provide a molecular platform for efficient protein–protein and protein–DNA interactions during DNA replication and repair. Once the repair is completed, the enzyme must stop the exonucleolytic process and switch to the polymerase mode. However, the cue to stop the degradation is unclear. We constructed several PCNA mutants and found that the exonuclease reaction was enhanced in the mutants lacking the conserved basic patch, located on the inside surface of PCNA. These mutants may mimic the Pol/PCNA complex processing the mismatched DNA, in which PCNA cannot interact rigidly with the irregularly distributed phosphate groups outside the dsDNA. Indeed, the exonuclease reaction with the wild type PCNA was facilitated by mismatched DNA substrates. PCNA may suppress the exonuclease reaction after the removal of the mismatched nucleotide. PCNA seems to act as a “brake” that stops the exonuclease mode of the DNA polymerase after the removal of a mismatched nucleotide from the substrate DNA, for the prompt switch to the DNA polymerase mode.

Biochemical Analysis of the Human Mismatch Repair Proteins hMutSα MSH2G674A-MSH6 and MSH2-MSH6T1219D

The heterodimeric human MSH2-MSH6 protein initiates DNA mismatch repair (MMR) by recognizing mismatched bases that result from replication errors. Msh2(G674A) or Msh6(T1217D) mice that have mutations in or near the ATP binding site of MSH2 or ATP hydrolysis catalytic site of MSH6 develop cancer and have a reduced lifespan due to loss of the MMR pathway (Lin, D. P., Wang, Y., Scherer, S. J., Clark, A. B., Yang, K., Avdievich, E., Jin, B., Werling, U., Parris, T., Kurihara, N., Umar, A., Kucherlapati, R., Lipkin, M., Kunkel, T. A., and Edelmann, W. (2004) Cancer Res. 64, 517-522; Yang, G., Scherer, S. J., Shell, S. S., Yang, K., Kim, M., Lipkin, M., Kucherlapati, R., Kolodner, R. D., and Edelmann, W. (2004) Cancer Cell 6, 139-150). Mouse embryonic fibroblasts from these mice retain an apoptotic response to DNA damage. Mutant human MutSα proteins MSH2(G674A)-MSH6(wt) and MSH2(wt)-MSH6(T1219D) are profiled in a variety of functional assays and as expected fail to support MMR in vitro, although they retain mismatch recognition activity. Kinetic analyses of DNA binding and ATPase activities and examination of the excision step of MMR reveal that the two mutants differ in their underlying molecular defects. MSH2(wt)-MSH6(T1219D) fails to couple nucleotide binding and mismatch recognition, whereas MSH2(G674A)-MSH6(wt) has a partial defect in nucleotide binding. Nevertheless, both mutant proteins remain bound to the mismatch and fail to promote efficient excision thereby inhibiting MMR in vitro in a dominant manner. Implications of these findings for MMR and DNA damage signaling by MMR proteins are discussed.

Single Molecule FRET Study of Nucleotide Binding Effects on Muts Proteins

The DNA mismatch repair (MMR) system is an essential component of the cellular DNA replication process that ensures high overall fidelity. The protein MutS initiates MMR by binding specifically to base-base mismatches and insertion-deletion mismatches in double stranded DNA. The active MutS dimer includes two ATP binding sites that hydrolyze ATP in both DNA bound and free states. ATP is also hydrolyzed as part of the MMR cascade involving MutS. Biochemical characterizations have shown that distinct nucleotides produce different interactions between MutS and mismatched DNA substrates, but the details of the conformations of MutS-mismatched DNA complexes under exposure to different nucleotides are not well known. We used single molecule fluorescence resonance energy transfer (smFRET) to study MutS from Thermus aquaticus in isolation and in complex with mismatched DNA substrates when exposed to a variety of different nucleotide conditions. We report results using intramolecular FRET from MutS or the DNA substrate as well as intermolecular FRET between MutS and the DNA. Our results allow MutS-DNA conformations resulting from specific nucleotide bound states to be characterized. We suggest possible roles for ATP cycling that could regulate the function of MutS in DNA MMR.

Characterization of physical and functional interactions between eukaryote-like Orc1/Cdc6 proteins and Y-family DNA polymerase in the hyperthermophilic archaeon Sulfolobus solfataricus

The roles of Y-family DNA polymerases and the regulation mechanisms are not well defined in Archaea. In this study, we performed in vitro and in vivo characterization of the physical interaction between the archaeon Sulfolobus solfataricus Y-family DNA polymerase (SsoPolY) and three eukaryote-like Orc1/Cdc6 proteins (SsoCdc6-1, SsoCdc6-2, and SsoCdc6-3). The effect of SsoCdc6-2 was the strongest, and the three SsoCdc6 proteins were shown to have very different effects on the function of SsoPolY. SsoCdc6-2 inhibited both the DNA-binding activity and DNA polymerization activity of SsoPolY on the DNA substrates containing mismatched bases, while it formed a large complex with SsoPolY and stimulated DNA-binding activity on paired primer-template DNA substrates. SsoCdc6-2 and S. solfataricus PCNA (SsoPCNA) showed a cooperative effect on polymerization by SsoPolY on paired DNA templates, but SsoCdc6 reduced the stimulating effect of SsoPCNA on this polymerization on mismatched DNA substrates. Therefore, we uncovered a DNA substrate-dependent SsoCdc6/SsoPolY interaction mechanism. This is the first evidence for a physical and functional linkage between archaeal eukaryote-like Orc1/Cdc6 proteins and Y-family DNA polymerase.

An insight into the biological functions of family X-DNA polymerase in DNA replication and repair of plant genome

Recently we have reported the characterization of a novel single subunit 62-kDa polypeptide with ddNTP-sensitive DNA polymerase activity from the developing seeds of mungbean (Vigna radiata). The protein showed higher expression and activity level during nuclear endoreduplication stages of mungbean seeds and similarity with mammalian DNA polymerase in many physicochemical properties.1 The enzyme was found to specifically interact with PCNA (proliferating cell nuclear antigen),2 and expressed in both meristematic and meiotic tissues. Functional assays have demonstrated binding of the enzyme to normal and mismatched DNA substrates and with fidelity DNA synthesis in moderately processive mode, suggesting probable involvement of the enzyme in both replication and recombination.3 Here we have discussed the position of mungbean DNA polymerase as a homologue of DNA Pol λone of the newly identified member of family-X DNA polymerase in plants and illustrated the functional relevance of this enzyme in maintaining the coordination between DNA replication and repair in plant genome.

Directed DNA Polymerase Evolution: Effects of Mutations in Motif C on the Mismatch‐Extension Selectivity of Thermus aquaticus DNA Polymerase

The selectivity of DNA polymerases for processing the canonical nucleotide and DNA substrate in favor of the noncanonical ones is the key to the integrity of the genome of every living species and to many biotechnological applications. The inborn ability of most DNA polymerases to abort efficient extension of mismatched DNA substrates adds to the overall DNA polymerase selectivity. DNA polymerases have been grouped into families according to their sequence. Within family A DNA polymerases, six motifs that come into contact with the substrates and form the active site have been discovered to be evolutionary highly conserved. Here we present results obtained from amino acid randomization within one motif, motif C, of thermostable Thermus aquaticus DNA polymerase. We have identified several distinct mutation patterns that increase the selectivity of mismatch extension. These results might lead to direct applications such as allele-specific PCR, as demonstrated by real-time PCR experiments and add to our understanding of DNA polymerase selectivity.

Preparation of DNA Substrates for In Vitro Mismatch Repair

Analyses in vitro of correction of DNA mismatches have been pivotal in biochemical dissection of mismatch repair pathways. However, the complex procedures needed to prepare DNA substrates for mismatch repair have posed substantial barriers to investigators who wish to pursue such analyses. Here we describe a simple, efficient way to prepare a variety of mismatched DNA substrates. We use in our procedure high-copy-number pUC19-derived plasmids, and a newly commercially available endonuclease N.BstNBI that makes site-specific single-strand nicks. The ability to prepare large substrate quantities in a relatively short time and to construct wider ranges of different mismatches in various sequence contexts will facilitate future research.

Recognition of sequence-directed DNA structure by the Klenow fragment of DNA polymerase I.

Time-resolved fluorescence spectroscopy was used to investigate the influence of sequence-directed DNA structure upon the interaction between the Klenow fragment of DNA polymerase I and a series of defined oligonucleotide primer/templates. 17/27-mer (primer/template) oligonucleotides containing a dansyl fluorophore conjugated to a modified deoxyuridine residue within the primer strand were used as substrates for binding to Klenow fragment. The time-resolved fluorescence anisotropy decay of the dansyl probe was analyzed in terms of two local environments, either solvent-exposed or buried, corresponding to primer/templates positioned with the primer 3' terminus in the polymerase site or the 3'-5' exonuclease site of the enzyme, respectively. Equilibrium constants for partitioning of DNA between the two sites were evaluated from the anisotropy decay data for primer/templates having different (A + T)-rich sequences flanking the primer 3' terminus. Primer/templates with AAAATG/TTTTAC and CGATAT/GCTATA terminal sequences (the nucleotides on the left refer to the last six bases at the 3' end of the primer, and the nucleotides on the right are the corresponding bases in the template) were bound mostly at the polymerase site. The introduction of single mismatches opposite the primer 3' terminus of these DNA substrates increased their partitioning into the 3'-5' exonuclease site, in accord with the results of an earlier study [Carver, T.E., Hochstrasser, R.A., and Millar, D.P. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 10670-10674]. In contrast, a primer/template with the terminal sequence CAATTT/GTTAAA, containing an A-tract element AATTT, exhibited a surprising preference for binding at the 3'-5' exonuclease site, despite the absence of mismatched bases in the DNA substrate. Interruption of the A-tract with a single AG step, to give the terminal sequence CAGTTT/GTCAAA, reversed the effect of the A-tract, causing the DNA to partition in favor of the polymerase site. Moreover, the presence of a single mismatch opposite the primer 3' terminus was also sufficient to reverse the effect of the A-tract, resulting in a distribution of DNA between polymerase and 3'-5' exonuclease sites that was similar to that observed for the other mismatched DNA substrates. Taken together, these results suggest that the A-tract adopts an unusual conformation that is disruptive to binding at the polymerase site. The effect of the A-tract on binding of DNA to the polymerase site is discussed in terms of the unusual helix structural parameters associated with these sequence elements and the difference between the local geometry of the A-tract and the conformation adopted by duplex DNA within the polymerase cleft. The results of this study show that in addition to base mismatches, Klenow fragment can also recognize irregularities in the helix geometry of perfectly base-paired DNA.

Effects of mutations on the partitioning of DNA substrates between the polymerase and 3'-5' exonuclease sites of DNA polymerase I (Klenow fragment).

Site-directed mutagenesis and time-resolved fluorescence spectroscopy were used to evaluate the contributions of individual amino acid side chains to the binding of DNA primer-templates to the 3'-5' exonuclease site of the large proteolytic fragment (Klenow fragment) of DNA polymerase I. Mutations were introduced into side chains that have been shown crystallographically to be in close proximity to a DNA 3' terminus bound at the 3'-5' exonuclease site. The wild-type residues were replaced by alanine in each case. To assess the effects of the mutations on DNA binding, time-resolved fluorescence anisotropy measurements were performed on dansyl-labeled primer-templates bound to the mutant enzymes. In contrast to techniques that simply monitor the overall binding of proteins to DNA, the time-resolved fluorescence anisotropy technique was used to determine the fractional occupancies of the polymerase and 3'-5' exonuclease active sites of Klenow fragment. Equilibrium constants describing the partitioning of DNA between the two active sites were obtained for nine different mutant enzymes bound to both matched and mismatched DNA sequences. Mutations of Leu361 and Phe473 caused the largest effects, significantly destabilizing the binding of mismatched DNA substrates to the 3'-5' exonuclease site relative to DNA bound at the polymerase site, consistent with structural data showing that the side chains of these residues are involved in intimate hydrophobic interactions with the 3' terminal and penultimate bases of the primer strand [Beese, L., and Steitz, T. A. (1991) EMBO J. 10, 25-33]. Mutations of the His660 and Glu357 side chains also resulted in significant effects on the binding of mismatched DNA to the 3'-5' exonuclease site. Surprisingly, mutation of Tyr497 increased the partitioning of mismatched DNA into the 3'-5' exonuclease site, suggesting that the tyrosine side chain in the wildtype enzyme destabilizes substrate binding, despite crystallographic data showing that Tyr497 is H-bonded to the DNA substrate. The effects of mutating the amino acid side chains that serve as ligands to two divalent metal ions bound at the 3'-5' exonuclease site, designated A and B, indicated that metal A also helps to bind DNA to the 3'-5' exonuclease site. These results demonstrate that the time-resolved fluorescence anisotropy technique can be used to quantify the energetic contributions associated with each of the crystallographically defined DNA-protein contacts at the 3'-5' exonuclease site.

Inhibition of human DNA ligase I activity by zinc and cadmium and the fidelity of ligation

Heavy metals, including zinc (Zn) and cadmium (Cd), are potentially important genotoxic agents in our environment. Here we report that human DNA ligase I, the major form of the enzyme in replicative cells, is a target for Zn and Cd ions. ZnCl2 at 0.8 mM caused complete inhibition of DNA ligase I activity, whereas only 0.04 mM CdCl2 was required to achieve a similar effect. Both metals affected all three steps of the reaction, namely, the formation of ligase-AMP intermediate, the transfer of the AMP to DNA and the ligation reaction that succeeds the formation of the AMP-DNA complex. Unlike F-ara-ATP and the natural protein inhibitor of DNA ligase-I, these metals may affect different domains of the enzyme. Moreover, these metal ions did not increase the rate of misligation of F-ara-A-modified DNA or mismatched DNA substrates, but considerable misligation was observed for the T:C mispairing. These data support the notion of high fidelity of the human DNA ligases and that the major action of these metal ions on the enzyme is their inhibitory function.

DNA determinants and substrate specificities of Escherichia coli MutY.

Potential DNA contacts involved in the specific interaction between the Escherichia coli MutY protein and a 40-mer oligonucleotide containing an A/G mismatch have been examined by alkylation interference techniques. Ethylation interference patterns suggest that more than five phosphates are involved in electrostatic interactions between MutY and DNA. Interestingly, MutY has more contacts on the G-strand than on the A-strand. Methylation at both the N-7 position of the mismatched G and the N-3 position of the mispaired A interfere with MutY binding. In addition to these mismatched bases, MutY also contacts purines on both sides of the mismatch. Binding and endonuclease activities of MutY were assayed with 20-mer oligonucleotides containing A/G, A/C, A/7,8-dihydro-8-oxo-guanine (A/GO), A/inosine (A/I), A/2-aminopurine (A/2AP), nebularine/G (N/G), inosine/G (I/G), 2AP/G, and 7-deaza-adenosine/G (Z/G) mispairs. The C-8 keto group of GO in A/GO contributes to a much tighter binding but weaker endonuclease activity than is seen with A/G. Because A/I is not specifically well recognized by MutY, the 2-amino group of G in A/G is essential for recognition. The C-6 keto group present in A/G but absent in A/2AP is also important for recognition. The 6-amino group of adenine appears not to be required for either binding or endonuclease activity because N/G is as good a substrate as A/G. The 2AP/G mispair is bound and cleaved weaker than is the A/G mispair. Binding and endonuclease activities are abolished when the N-7 group of A is replaced by C-7 as in the Z/G mispair. When a C-6 keto group is present as in the I/G pair, its binding by MutY is as good as for A/G, but no endonuclease activity is observed. Taken together, our data suggest that DNA sequences proximal to and specific functional groups of mismatched bases are necessary for recognition and catalysis by MutY protein.