Asymmetric ATP Binding and Hydrolysis Activity of the \u3cem\u3eThermus aquaticus\u3c/em\u3e MutS Dimer Is Key to Modulation of Its Interactions with Mismatched DNA

Prokaryotic MutS and eukaryotic Msh proteins recognize base pair mismatches and insertions or deletions in DNA and initiate mismatch repair. These proteins function as dimers (and perhaps higher order oligomers) and possess an ATPase activity that is essential for DNA repair. Previous studies of Escherichia coli MutS and eukaryotic Msh2−Msh6 proteins have revealed asymmetry within the dimer with respect to both DNA binding and ATPase activities. We have found the Thermus aquaticus MutS protein amenable to detailed investigation of the nature and role of this asymmetry. Here, we show that (a) in a MutS dimer one subunit (S1) binds nucleotide with high affinity and the other (S2) with 10-fold weaker affinity, (b) S1 hydrolyzes ATP rapidly while S2 hydrolyzes ATP at a 30−50-fold slower rate, (c) mismatched DNA binding to MutS inhibits ATP hydrolysis at S1 but slow hydrolysis continues at S2, and (d) interaction between mismatched DNA and MutS is weakened when both subunits are occupied by ATP but remains stable when S1 is occupied by ATP and S2 by ADP. These results reveal key MutS species in the ATPase pathway; S1ADP−S2ATP is formed preferentially in the absence of DNA or in the presence of fully matched DNA, while S1ATP−S2ATP and S1ATP−S2ADP are formed preferentially in the presence of mismatched DNA. These MutS species exhibit differences in interaction with mismatched DNA that are likely important for the mechanism of MutS action in DNA repair

Mismatch Recognition-Coupled Stabilization of Msh2-Msh6 in an ATP-Bound State at the Initiation of DNA Repair

Mismatch repair proteins correct errors in DNA via an ATP-driven process. In eukaryotes, the Msh2-Msh6 complex recognizes base pair mismatches and small insertion/deletions in DNA and initiates repair. Both Msh2 and Msh6 proteins contain Walker ATP-binding motifs that are necessary for repair activity. To understand how these proteins couple ATP binding and hydrolysis to DNA binding/mismatch recognition, the ATPase activity of Saccharomyces cerevisiae Msh2-Msh6 was examined under pre-steady-state conditions. Acid-quench experiments revealed that in the absence of DNA, Msh2-Msh6 hydrolyzes ATP rapidly (burst rate = 3 s-1 at 20 °C) and then undergoes a slow step in the pathway that limits catalytic turnover (kcat = 0.1 s-1). ATP is hydrolyzed similarly in the presence of fully matched duplex DNA; however, in the presence of a G:T mismatch or +T insertion-containing DNA, ATP hydrolysis is severely suppressed (rate = 0.1 s-1). Pulse-chase experiments revealed that Msh2-Msh6 binds ATP rapidly in the absence or in the presence of DNA (rate = 0.1 μM-1 s-1), indicating that for the Msh2-Msh6·mismatched DNA complex, a step after ATP binding but before or at ATP hydrolysis is the rate-limiting step in the pathway. Thus, mismatch recognition is coupled to a dramatic increase in the residence time of ATP on Msh2-Msh6. This mismatch-induced, stable ATP-bound state of Msh2-Msh6 likely signals downstream events in the repair pathway

Role of a Conserved Glutamate Residue in the \u3cem\u3eEscherichia coli\u3c/em\u3e SecA ATPase Mechanism

Escherichia coli SecA uses ATP to drive the transport of proteins across cell membranes. Glutamate 210 in the “DEVD” Walker B motif of the SecA ATP-binding site has been proposed as the catalytic base for ATP hydrolysis (Hunt, J. F., Weinkauf, S., Henry, L., Fak, J. J., McNicholas, P., Oliver, D. B., and Deisenhofer, J. (2002) Science 297, 2018–2026). Consistent with this hypothesis, we find that mutation of glutamate 210 to aspartate results in a 90-fold reduction of the ATP hydrolysis rate compared with wild type SecA, 0.3 s–1versus 27 s–1, respectively. SecA-E210D also releases ADP at a slower rate compared with wild type SecA, suggesting that in addition to serving as the catalytic base, glutamate 210 might aid turnover as well. Our results contradict an earlier report that proposed aspartate 133 as the catalytic base (Sato, K., Mori, H., Yoshida, M., and Mizushima, S. (1996) J. Biol. Chem. 271, 17439–17444). Re-evaluation of the SecA-D133N mutant used in that study confirms its loss of ATPase and membrane translocation activities, but surprisingly, the analogous SecA-D133A mutant retains full activity, revealing that this residue does not play a key role in catalysis

Contribution of Msh2 and Msh6 Subunits to the Asymmetric ATPase and DNA Mismatch Binding Activities of \u3cem\u3eSaccharomyces cerevisiae\u3c/em\u3e Msh2–Msh6 Mismatch Repair Protein

Previous analyses of both Thermus aquaticus MutS homodimer and Saccharomyces cerevisiae Msh2–Msh6 heterodimer have revealed that the subunits in these protein complexes bind and hydrolyze ATP asymmetrically, emulating their asymmetric DNA binding properties. In the MutS homodimer, one subunit (S1) binds ATP with high affinity and hydrolyzes it rapidly, while the other subunit (S2) binds ATP with lower affinity and hydrolyzes it at an apparently slower rate. Interaction of MutS with mismatched DNA results in suppression of ATP hydrolysis at S1—but which of these subunits, S1 or S2, makes specific contact with the mismatch (e.g., base stacking by a conserved phenylalanine residue) remains unknown. In order to answer this question and to clarify the links between the DNA binding and ATPase activities of each subunit in the dimer, we made mutations in the ATPase sites of Msh2 and Msh6 and assessed their impact on the activity of the Msh2–Msh6 heterodimer (in Msh2–Msh6, only Msh6 makes base specific contact with the mismatch). The key findings are: (a) Msh6 hydrolyzes ATP rapidly, and thus resembles the S1 subunit of the MutS homodimer, (b) Msh2 hydrolyzes ATP at a slower rate, and thus resembles the S2 subunit of MutS, (c) though itself an apparently weak ATPase, Msh2 has a strong influence on the ATPase activity of Msh6, (d) Msh6 binding to mismatched DNA results in suppression of rapid ATP hydrolysis, revealing a “cis” linkage between its mismatch recognition and ATPase activities, (e) the resultant Msh2–Msh6 complex, with both subunits in the ATP-bound state, exhibits altered interactions with the mismatch

Overproduction and Analysis of Eukaryotic Multiprotein Complexes in \u3cem\u3eEscherichia coli\u3c/em\u3e Using a Dual-vector Strategy

Biochemical studies of eukaryotic proteins are often constrained by low availability of these typically large, multicomponent protein complexes in pure form. Escherichia coli is a commonly used host for large-scale protein production; however, its utility for eukaryotic protein production is limited because of problems associated with transcription, translation, and proper folding of proteins. Here we describe the development and testing of pLANT, a vector that addresses many of these problems simultaneously. The pLANT vector contains a T7 promoter-controlled expression unit, a p15A origin of replication, and genes for rare transfer RNAs and kanamycin resistance. Thus, the pLANT vector can be used in combination with the pET vector to coexpress multiple proteins in E. coli. Using this approach, we have successfully produced high-milligram quantities of two different Saccharomyces cerevisiae complexes in E. coli: the heterodimeric Msh2–Msh6 mismatch repair protein (248 kDa) and the five-subunit replication factor C clamp loader (250 kDa). Quantitative analyses indicate that these proteins are fully active, affirming the utility of pLANT+pET-based production of eukaryotic proteins in E. coli for in vitro studies of their structure and function

Graphene Patterned Microchip For Colorectal Cancer Detection

Cancer currently stands as the second-leading cause of death worldwide. Studies reveal colorectal cancer (CRC) to be the 4th leading cause of mortality due to cancer. It is estimated that about 30% of CRC cases are hereditary, of which 5% are attributed by known syndromes, particularly Lynch Syndrome. This pilot study aims to fabricate a DNA-graphene-polypyrrole (DGP) based biosensor to diagnose deficiency of functional MMR proteins present in patients at a scale of less than ng/ ml. We have followed LAB-on-CHIP method. We find that the interactive forces between avidin and graphene are mainly hydrophobic, along with some van der Waals, electrostatic and hydrogen bonding interactions. Different mismatch combinations were performed, to prove the activity of each component on the chip. 30 such combinations were done. Electrochemical impedance spectroscopy was done to confirm the working of the bio sensor by corresponding change in electrical impedance. To assist this real-world system, we have carried out simulation studies as well. In the simulation studies from 0-200ns, we present the progression structures of human MutS protein to biotinylated DNA that has been fixed to simulate the manner of a biosensor, furthermore the mismatch within the DNA has been manually introduced with the aid of computational tools to reveal the interactions of the DNA and the protein. This research additionally permits us in early detection of colorectal cancer and the mapping and expertise of the method related to the area of the mismatch repair

Functional Protein detection for DNA Mismatch Repair: A Novel Nano-biosensor for Cancer Diagnostics

Cancer currently stands as the second-leading cause of death worldwide. Studies reveal colorectal cancer (CRC) to be the 4th leading cause of mortality due to cancer. It is estimated that about 30% of CRC cases are hereditary, of which 5% are attributed by known syndromes, particularly Lynch Syndrome. Lynch Syndrome (LS) is caused by loss or malfunction of proteins responsible for DNA mismatch repair proteins (MMR), mostly MLH1 and MSH2, causing increased risks of developing CRC. Despite the small percentage accounted with the disease, the severity of the illness still remains immense since 80% of these patients eventually develop CRC and an overwhelming 40-60 % of female patients develop endometrial cancer, the major of cancer in women in developing nations. Current diagnostic procedures for LS involve testing tumor tissue for microsatellite instability and the presence/absence of MMR proteins by immunohistochemistry (IHC), followed by germine testing for mutations in MMR genes, if warranted. While genetic testing is becoming more cost-effective and accessible, a major problem with this approach is that the functional and pathological consequences of a majority of mutations and small insertions/deletions in MMR genes are unknown, rendering the tests results inconclusive in many cases. Therefore the need for an accurate means of early diagnosis is clearly imminent to prevent the growing threat. This pilot study aims to fabricate a DNA-graphene-polypyrole (DGP) based biosensor to diagnose deficiency of functional MMR proteins present in patients at a scale of less than ng/ ml. Biotinylated DNA probes are immobilized on an avidin film coated over a polypyrole-graphene layer, which in turn is finely deposited over a gold-plated circuit using electrochemical polymerization technique. A detailed morphological characterization has been performed using both scanning electron microscope (SEM) and atomic force microscope (AFM) topography of the DGP biosensor. Quantitative analysis is performed by measuring electrochemical impedance spectroscopy (EIS) where the change in impedance of the samples is recorded by varying frequency between 1MHz- 100 MHz. Though still a work in progress, we assume that the attachment of the MMR proteins onto the mismatched sequences should alter the impedance of the fabricated DGP biosensor. A final analysis is being conducted to confirm our assumptions

A Novel Nano-biosensor for Colorectal Cancer Diagnostics by Detecting DNA Mismatch Repair Proteins

Cancer currently stands as the second-leading cause of death worldwide. Studies reveal colorectal cancer (CRC) to be the 4th leading cause of mortality due to cancer. It is estimated that about 30% of CRC cases are hereditary, of which 5% are attributed by known syndromes, particularly Lynch Syndrome. Lynch Syndrome (LS) is caused by loss or malfunction of proteins responsible for DNA mismatch repair proteins (MMR), mostly MLH1 and MSH2, causing increased risks of developing CRC. Despite the small percentage accounted with the disease, the severity of the illness still remains immense since 80% of these patients eventually develop CRC and an overwhelming 40-60 % of female patients develop endometrial cancer, the major form of cancer in women in the developing nations. This pilot study aims to fabricate a DNA-graphene-polypyrole (DGP) based biosensor to diagnose deficiency of functional MMR proteins present in patients at a scale of less than ng/ ml. Fundamental understanding of interactions at the interface of biological molecules, such as proteins, and nanomaterials is therefore crucial for developing such biocompatible hybrid materials and biosensing platforms. Conductive nanomaterials-based biosensors offer the advantage of higher sensitivity and reliable diagnosis mainly due to their superior specific surface area and ballistic conductivity. Such films that immobilize proteins can synergize the properties of transducers and molecular recognition elements in order to improve biosensor performance and diversity. Here we report for the first time, using a combined molecular dynamics simulations and experimental approach, the interactions between avidin and a graphene surface, which is being developed as a sensing platform for early detection of DNA mismatch repair proteins. We find that the interactive forces between avidin and graphene are mainly hydrophobic, along with some van der Waals, electrostatic and hydrogen bonding interactions. Notably, the structure and function of the avidin molecule is preserved after its adsorption on the graphene surface. The MD results agree well with scanning electron microscopy (SEM) and electrochemical impedance spectroscopy (EIS) analysis of avidin immobilized on a graphenated polypyrrole (G-PPy) conductive substrate, which confirm adsorption of avidin on graphene nanoplatelets and corresponding changes in electrical impedance, respectively. A final analysis is being conducted to confirm our hypothesis

The ATPase mechanism of UvrA2 reveals the distinct roles of proximal and distal ATPase sites in nucleotide excision repair

The UvrA2 dimer finds lesions in DNA and initiates nucleotide excision repair. Each UvrA monomer contains two essential ATPase sites: proximal (P) and distal (D). The manner whereby their activities enable UvrA2 damage sensing and response remains to be clarified. We report three key findings from the first pre-steady state kinetic analysis of each site. Absent DNA, a P2ATP-D2ADP species accumulates when the low-affinity proximal sites bind ATP and enable rapid ATP hydrolysis and phosphate release by the highaffinity distal sites, and ADP release limits catalytic turnover. Native DNA stimulates ATP hydrolysis by all four sites, causing UvrA2 to transition through a different species, P2ADP-D2ADP. Lesion-containing DNA changes the mechanism again, suppressing ATP hydrolysis by the proximal sites while distal sites cycle through hydrolysis and ADP release, to populate proximal ATP-bound species, P2ATP-Dempty and P2ATPD2ATP. Thus, damaged and native DNA trigger distinct ATPase site activities, which could explain why UvrA2 forms stable complexes with UvrB on damaged DNA compared with weaker, more dynamic complexes on native DNA. Such specific coupling between the DNA substrate and the ATPase mechanism of each site provides new insights into how UvrA2 utilizes ATP for lesion search, recognition and repair

Missed cleavage opportunities by FEN1 lead to Okazaki fragment maturation via the long-flap pathway.

RNA-DNA hybrid primers synthesized by low fidelity DNA polymerase α to initiate eukaryotic lagging strand synthesis must be removed efficiently during Okazaki fragment (OF) maturation to complete DNA replication. In this process, each OF primer is displaced and the resulting 5'-single-stranded flap is cleaved by structure-specific 5'-nucleases, mainly Flap Endonuclease 1 (FEN1), to generate a ligatable nick. At least two models have been proposed to describe primer removal, namely short- and long-flap pathways that involve FEN1 or FEN1 along with Replication Protein A (RPA) and Dna2 helicase/nuclease, respectively. We addressed the question of pathway choice by studying the kinetic mechanism of FEN1 action on short- and long-flap DNA substrates. Using single molecule FRET and rapid quench-flow bulk cleavage assays, we showed that unlike short-flap substrates, which are bound, bent and cleaved within the first encounter between FEN1 and DNA, long-flap substrates can escape cleavage even after DNA binding and bending. Notably, FEN1 can access both substrates in the presence of RPA, but bending and cleavage of long-flap DNA is specifically inhibited. We propose that FEN1 attempts to process both short and long flaps, but occasional missed cleavage of the latter allows RPA binding and triggers the long-flap OF maturation pathway