Loader Starsat 2000 Hd
Macabeo Eastman
I see that you are using CCS 7.4. With this older version of CCS it is likely that you also have an older version of C2000 device support package and/or other components, and that is most probably contributing to the error.
If possible, I would recommend installing the latest CCS version 9.1.0 (which should already have these updated components). Or at a minimum install CCS 8.3 and make sure that the components mentioned in the referenced thread (C2000 device support, Debug Server flash and TI Emulator package) are all updated via CCS menu Help->Check for Updates.
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(A) Crystal structure (left) and schematic diagram (right) of the clamp loader. (B) The clamp loading cycle, from left to right, showing the key stages of loading the sliding clamp around primer-templated DNA. (C) Key elements of AAA+ modules, shown here at the interface between neighboring ATPase subunits at positions B and C with DNA and the sliding clamp.
All clamp-loader complexes consist of five subunits that have arisen through gene duplication of an ancestral ATPase subunit. The ATPase subunits of clamp loaders are members of the very large and diverse family of AAA+ ATPases, which are oligomeric proteins that bind and hydrolyze ATP at interfacial sites (Gates and Martin, 2020; Neuwald et al., 1999). Each subunit in a clamp-loader complex contains a two-domain AAA+ module that is characteristic of the AAA+ ATPases. The AAA+ module in each subunit is connected to a C-terminal collar domain that is responsible for oligomerization of the clamp loader (Bowman et al., 2004; Guenther et al., 1997; Jeruzalmi et al., 2001; Figure 1A). The five subunits of the clamp loader are referred to as the A, B, C, D, and E subunits, and the AAA+ modules within a complex form a spiral assembly when bound to ATP (Bowman et al., 2004). In some clamp loaders, the subunits at the A or E positions are degenerate and have lost the capacity to bind or hydrolyze ATP.
The base of each ATPase domain of a clamp-loader complex contains a loop that binds to a pocket on the surface of the sliding clamp (Bowman et al., 2004; Gulbis et al., 1996; Kelch et al., 2011). Pseudo-symmetry in the structure of the clamp allows all subunits of the clamp loader to engage a clamp simultaneously, using essentially the same set of interactions. Because of the spiral configuration of the ATP-bound AAA+ modules of the clamp loader, the interaction with the sliding clamp results in the conversion of the normally closed and planar clamp into an open spiral form (Kelch et al., 2011; Miyata et al., 2005). A key mechanism that enables the clamp loader to play a matchmaker role is the suppression, in the absence of DNA, of the ATPase activity of the clamp loader by the clamp (Turner et al., 1999). The recognition of primer-template junctions causes the rearrangement of the ATPase subunits into catalytically competent configurations. This triggers cooperative ATP hydrolysis, which weakens the spiral organization of the ATPase modules and causes the release of the closed clamp on DNA (Kelch et al., 2012).
The sequences of sliding clamps and clamp loaders display a high degree of variation across the different branches of life, and even within individual branches such as the T4-like bacteriophages. To what extent does the mutational sensitivity of the system in a particular organism, carrying out the essential function of DNA replication, reflect the sequence diversity seen across the spread of life? The clamp loader subunits respond cooperatively to the clamp, ATP and DNA. How do the mechanisms underlying this cooperativity impose constraints on the sequence? Are there residues that are highly sensitive to mutation, but do not have an obvious role in catalysis or molecular recognition? The depth of structural and biochemical information available for clamp-loader complexes make them particularly suitable for the application of deep-mutagenesis methods aimed at addressing these questions (Boucher et al., 2016; Boucher et al., 2014; Fowler and Fields, 2014; Shah and Kuriyan, 2019).
(A) Schematic depicts the generation of T4del. The clamp-loader locus in wild-type T4 bacteriophage, containing genes for the sliding clamp and the ATPase and clasp subunits of the clamp loader, is replaced with a CRISPR-cas12 target site. See methods for details. (B) In the high-throughput phage-propagation assay, T4del infects bacteria carrying (i) a plasmid-encoded CRISPR-cas12 (not shown) programmed to target the recombination site inserted in A and (ii) a plasmid containing variant genes of the clamp-loader locus. Upon infection, the clamp-loader locus recombines into the T4del genome, and this genome is replicated by the variant of the clamp and clamp-loader genes present in each cell. Variants with reduction in function in the clamp-loader activity will produce fewer phage particles relative to the variants with wildtype-like activity. (C) Workflow for comprehensive assessment of fitness effects of all possible single amino-acid mutants of the sliding clamp and the clamp-loader subunits. Codons corresponding to each amino acid are individually mutagenized to NNS (N is a mixture of all four nucleotide bases, S is a mixture of G and C) by PCR, combined in equimolar ratios and transformed into E. coli for cloning.
(A) The location of the 10-residue region in the structure of the clamp loader. (B) Relative fitness values of all 32 codons from the NNS substitutions at mutationally sensitive positions (107 and 111) and mutationally tolerant positions (112 and 115). (C) Relative fitness values for each amino acid substitution in the 10-residue region depicted as a heatmap, with wildtype-like fitness as white (score of 0), loss of function as shades of blue and gain of function as shades of red. Pixels corresponding to the amino acid present in the wild-type sequence at each position are outlined in black. Sequence logo generated from 1000 related phage ATPase sequences, with the sequence of T4 bacteriophage shown above the heatmap for reference. (D) Agreement between fitness measurements from three replicate experiments, where each point in the scatter plot represents the fitness measurements made from two trials.
(A) ATP hydrolysis rates of clamp loader variants with point mutations in the ATPase subunit, measured by a coupled-kinase assay (Goedken et al., 2005) (see Materials and methods for details). (B) DNA replication assay (Seville et al., 1996) performed with M13 phage genomic ssDNA as template. -DNA Pol. refers to the reaction setup with all the necessary proteins from wild-type bacteriophage T4 except the T4 DNA polymerase. DNA replication with the Phusion DNA polymerase system (ThermoFisher), with and without the DNA primer, sets the expected dynamic range of the assay.
Residues within the ten-residue segment that are expected to be functionally critical are highly sensitive to mutation, providing additional reassurance that the assay is reporting faithfully on replication fitness. The first four residues of this segment (residues 107 to 110, with sequence DEFD) is part of the Walker B motif, and corresponds to the DExD/H motif that is found in many helicases (this motif is DExD in eukaryotic, viral, and archaeal clamp loaders). Asp 107 and Glu 108, at the first two positions of the DExD motif, coordinate the Mg2+ ion bound to ATP. Glu 108 has an additional catalytic role in activating the water molecule that attacks the terminal phosphate group of ATP (Ogura and Wilkinson, 2001). Consistent with their crucial functions, these two residues do not tolerate any substitution in the phage-propagation assay. Even the conservative substitutions of Asp 107 by glutamic acid, or Glu 108 by aspartic acid, result in fitness scores corresponding to at least a 200-fold reduction in the number of output phage.
Asp 110, the fourth residue in the DExD motif, is likely to play a regulatory rather than a catalytic role. Asp 110 forms an ion pair with the sidechain of Arg 122 in the adjacent subunit when the clamp loader is bound to DNA (Kelch et al., 2011), but this interaction is disrupted in a way that blocks ATP hydrolysis when DNA is not bound, an important point that we return to later (Bowman et al., 2004; Gaubitz et al., 2020). The mutational data in Figure 3 show that Asp 110 is highly sensitive to mutation, with only Asp and Glu being tolerated. Arg 111 is a DNA-interacting residue that is extremely sensitive to any substitution in the phage replication assay. Even the conservative replacement of this arginine by lysine results in a greater than 200-fold decrease in phage propagation.
We purified six mutant clamp loaders and carried out biochemical assays to assess the effect of the mutations on function. The six variants that were analyzed had fitness scores spanning the range of observed values. Biochemical assays cannot account for the complexity of phage propagation, and so they are potentially limited in their ability to validate the results of the phage-propagation assay. Nevertheless, our experiments suggest that a reduction of fitness in the phage-propagation assays corresponds to a loss of efficiency in the clamp-loading reaction and in phage replication.
Figure 5 shows a heatmap corresponding to results of the deep mutagenesis analysis for the AAA+ module of the T4 clamp loader. For comparison, the sequence-conservation profile for the AAA+ modules of a set of bacteriophage clamp loaders is shown above the heat map. We generated this conservation profile by aligning 1000 sequences of clamp-loader ATPase subunits from bacteriophage genomes, and the results are represented as a sequence logo (Crooks et al., 2004) in Figure 5. The height of the single-letter codes for an amino acid at a particular position in the sequence logo is proportional to the sequence conservation at that position.
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