Biomedical subjects

Aaron Johnson

Publications and source records attributed to Aaron Johnson.

9 recordsLinked to original sources

PubMed2006

The replication factor C clamp loader requires arginine finger sensors to drive DNA binding and proliferating cell nuclear antigen loading.

Replication factor C (RFC) is an AAA+ heteropentamer that couples the energy of ATP binding and hydrolysis to the loading of the DNA polymerase processivity clamp, proliferating cell nuclear antigen (PCNA), onto DNA. RFC consists of five subunits in a spiral arrangement (RFC-A, -B, -C, -D, and -E, corresponding to subunits RFC1, RFC4, RFC3, RFC2, and RFC5, respectively). The RFC subunits are AAA+ family proteins and the complex contains four ATP sites (sites A, B, C, and D) located at subunit interfaces. In each ATP site, an arginine residue from one subunit is located near the gamma-phosphate of ATP bound in the adjacent subunit. These arginines act as "arginine fingers" that can potentially perform two functions: sensing that ATP is bound and catalyzing ATP hydrolysis. In this study, the arginine fingers in RFC were mutated to examine the steps in the PCNA loading mechanism that occur after RFC binds ATP. This report finds that the ATP sites of RFC function in distinct steps during loading of PCNA onto DNA. ATP binding to RFC powers recruitment and opening of PCNA and activates a gamma-phosphate sensor in ATP site C that promotes DNA association. ATP hydrolysis in site D is uniquely stimulated by PCNA, and we propose that this event is coupled to PCNA closure around DNA, which starts an ordered hydrolysis around the ring. PCNA closure severs contact to RFC subunits D and E (RFC2 and RFC5), and the gamma-phosphate sensor of ATP site C is switched off, resulting in low affinity of RFC for DNA and ejection of RFC from the site of PCNA loading.

Adenosine Triphosphate↗

PubMed2006

Mechanism of proliferating cell nuclear antigen clamp opening by replication factor C.

The eukaryotic replication factor C (RFC) clamp loader is an AAA+ spiral-shaped heteropentamer that opens and closes the circular proliferating cell nuclear antigen (PCNA) clamp processivity factor on DNA. In this study, we examined the roles of individual RFC subunits in opening the PCNA clamp. Interestingly, Rfc1, which occupies the position analogous to the delta clamp-opening subunit in the Escherichia coli clamp loader, is not required to open PCNA. The Rfc5 subunit is required to open PCNA. Consistent with this result, Rfc2.3.4.5 and Rfc2.5 subassemblies are capable of opening and unloading PCNA from circular DNA. Rfc5 is positioned opposite the PCNA interface from Rfc1, and therefore, its action with Rfc2 in opening PCNA indicates that PCNA is opened from the opposite side of the interface that the E. coli delta wrench acts upon. This marks a significant departure in the mechanism of eukaryotic and prokaryotic clamp loaders. Interestingly, the Rad.RFC DNA damage checkpoint clamp loader unloads PCNA clamps from DNA. We propose that Rad.RFC may clear PCNA from DNA to facilitate shutdown of replication in the face of DNA damage.

Chromatography, Gel↗

PubMed2005

DNA ligase: getting a grip to seal the deal.

The crystal structure of human DNA ligase I catches the enzyme just before the last step of ligation and shows that the protein wraps completely around nicked DNA. The elegant structure explains how ligase attains fidelity for the sealing operation.

DNA Ligase ATP↗

PubMed2005

Cellular DNA replicases: components and dynamics at the replication fork.

DNA replicases are multicomponent machines that have evolved clever strategies to perform their function. Although the structure of DNA is elegant in its simplicity, the job of duplicating it is far from simple. At the heart of the replicase machinery is a heteropentameric AAA+ clamp-loading machine that couples ATP hydrolysis to load circular clamp proteins onto DNA. The clamps encircle DNA and hold polymerases to the template for processive action. Clamp-loader and sliding clamp structures have been solved in both prokaryotic and eukaryotic systems. The heteropentameric clamp loaders are circular oligomers, reflecting the circular shape of their respective clamp substrates. Clamps and clamp loaders also function in other DNA metabolic processes, including repair, checkpoint mechanisms, and cell cycle progression. Twin polymerases and clamps coordinate their actions with a clamp loader and yet other proteins to form a replisome machine that advances the replication fork.

DNA Replication↗

PubMed2004

Disappearing "T1 black holes" in an animal model of multiple sclerosis.

Brain MRI in multiple sclerosis (MS) frequently shows areas of hypointensity in the white matter on T1 weighted sequences ("T1 black holes"). These areas are thought to be consistent with irreversible axonal loss. In this study T1 black holes were characterized in Theiler's Murine Encephalitis Virus infection, an established model of demyelinating diseases in mice. The spectrum of TMEV is broad in different strains. C57BL/6J mice develop a self-limited brain disease, which resolves within 4-6 weeks. We followed six mice with serial MRI and MRS on days 0, 3,7,21 and 45. The studies were performed in a 7 Tesla magnet. Periventricular and parahippocampal T1 black holes seen as early as 3 days, with decreasing NAA/Cre ratio on MRS. The extent of pathology was most severe on days 3 and 7. T1 black holes are thought to be consistent with areas of irreversible axonal loss. This is challenged by our observations of resolution of T1 black holes by day 45. This was concomitant with the normalization of MRS findings in the areas of interest. We conclude that T1 black holes may represent a transient phenomenon in this model of MS. The recovery of these areas studied suggests an active repair mechanism.

Animals↗

PubMed2004

Fluorescence measurements on the E.coli DNA polymerase clamp loader: implications for conformational changes during ATP and clamp binding.

Sliding clamps are ring-shaped proteins that tether DNA polymerases to their templates during processive DNA replication. The action of ATP-dependent clamp loader complexes is required to open the circular clamps and to load them onto DNA. The crystal structure of the pentameric clamp loader complex from Escherichia coli (the gamma complex), determined in the absence of nucleotides, revealed a highly asymmetric and extended form of the clamp loader. Consideration of this structure suggested that a compact and more symmetrical inactive form may predominate in solution in the absence of crystal packing forces. This model has the N-terminal domains of the delta and delta' subunits of the clamp loader close to each other in the inactive state, with the clamp loader opening in a crab-claw-like fashion upon ATP-binding. We have used fluorescence resonance energy transfer (FRET) to investigate the structural changes in the E.coli clamp loader complex that result from ATP-binding and interactions between the clamp loader and the beta clamp. FRET measurements using fluorophores placed in the N-terminal domains of the delta and delta' subunits indicate that the distances between these subunits in solution are consistent with the previously crystallized extended form of the clamp loader. Furthermore, the addition of nucleotide and clamp to the labeled clamp loader does not appreciably alter these FRET distances. Our results suggest that the changes that occur in the relative positioning of the delta and delta' subunits when ATP binds to and activates the complex are subtle, and that crab-claw-like movements are not a significant component of the clamp loader mechanism.

Adenosine Triphosphate↗

PubMed2003

In vivo magnetic resonance imaging of immune cells in the central nervous system with superparamagnetic antibodies.

We developed a novel MRI technique to image immune cell location and homing in vivo to the central nervous system (CNS). Superparamagnetic antibodies specific for cell surface markers allowed imaging of CD4+ T cells, CD8+ T cells, and Mac1+ cells in the CNS of mice infected with Theiler's murine encephalomyelitis virus (TMEV) and in mice with experimental autoimmune encephalomyelitis (EAE). Superparamagnetic antibodies have excellent T2, T2*, and good T1 relaxation properties, which makes them ideal MRI contrast materials. Immunohistochemistry of corresponding sections confirmed the specificity of the technique to detect immune cell types in the CNS. This powerful technique has potential to image any cell with unique surface antigens. Because superparamagnetic antibodies similar to those used in the study are approved for human use, the in vivo MRI technique we have described could be developed for human use.

Animals↗

PubMed2003

Mechanism of loading the Escherichia coli DNA polymerase III sliding clamp: II. Uncoupling the beta and DNA binding activities of the gamma complex.

Sliding clamps tether DNA polymerases to DNA to increase the processivity of synthesis. The Escherichia coli gamma complex loads the beta sliding clamp onto DNA in an ATP-dependent reaction in which ATP binding and hydrolysis modulate the affinity of the gamma complex for beta and DNA. This is the second of two reports (Williams, C. R., Snyder, A. K., Kuzmic, P., O'Donnell, M., and Bloom, L. B. (2004) J. Biol. Chem. 279, 4376-4385) addressing the question of how ATP binding and hydrolysis regulate specific interactions with DNA and beta. Mutations were made to an Arg residue in a conserved SRC motif in the delta' and gamma subunits that interacts with the ATP site of the neighboring gamma subunit. Mutation of the delta' subunit reduced the ATP-dependent beta binding activity, whereas mutation of the gamma subunits reduced the DNA binding activity of the gamma complex. The gamma complex containing the delta' mutation gave a pre-steady-state burst of ATP hydrolysis, but at a reduced rate and amplitude relative to the wild-type gamma complex. A pre-steady-state burst of ATP hydrolysis was not observed for the complex containing the gamma mutations, consistent with the reduced DNA binding activity of this complex. The differential effects of these mutations suggest that ATP binding at the gamma1 site may be coupled to conformational changes that largely modulate interactions with beta, whereas ATP binding at the gamma2 and/or gamma3 site may be coupled to conformational changes that have a major role in interactions with DNA. Additionally, these results show that the "arginine fingers" play a structural role in facilitating the formation of a conformation that has high affinity for beta and DNA.

Adenosine Triphosphate↗

PubMed2003

Ordered ATP hydrolysis in the gamma complex clamp loader AAA+ machine.

The gamma complex couples ATP hydrolysis to the loading of beta sliding clamps onto DNA for processive replication. The gamma complex structure shows that the clamp loader subunits are arranged as a circular heteropentamer. The three gamma motor subunits bind ATP, the delta wrench opens the beta ring, and the delta' stator modulates the delta-beta interaction. Neither delta nor delta' bind ATP. This report demonstrates that the delta' stator contributes a catalytic arginine for hydrolysis of ATP bound to the adjacent gamma(1) subunit. Thus, the delta' stator contributes to the motor function of the gamma trimer. Mutation of arginine 169 of gamma, which removes the catalytic arginines from only the gamma(2) and gamma(3) ATP sites, abolishes ATPase activity even though ATP site 1 is intact and all three sites are filled. This result implies that hydrolysis of the three ATP molecules occurs in a particular order, the reverse of ATP binding, where ATP in site 1 is not hydrolyzed until ATP in sites 2 and/or 3 is hydrolyzed. Implications of these results to clamp loaders of other systems are discussed.

Adenosine Triphosphatases↗