We report here on the genome sequence of Pasteurella multocida Razi 0002 of avian origin, isolated in Iran. The genome has a size of 2,289,036 bp, a GC content of 40.3%, and is predicted to contain 2,079 coding sequences.

No abstract

DNA helicases are essential motor proteins that function to unwind duplex DNA to yield the transient single-stranded DNA intermediates required for replication, recombination, and repair. These enzymes unwind duplex DNA and translocate along DNA in reactions that are coupled to the binding and hydrolysis of 5'-nucleoside triphosphates (NTP). Although these enzymes are essential for DNA metabolism, the molecular details of their mechanisms are only beginning to emerge. This review discusses mechanistic aspects of helicase-catalyzed DNA unwinding and translocation with a focus on energetic (thermodynamic), kinetic, and structural studies of the few DNA helicases for which such information is available. Recent studies of DNA and NTP binding and DNA unwinding by the Escherichia coli (E. coli) Rep helicase suggest that the Rep helicase dimer unwinds DNA by an active, rolling mechanism. In fact, DNA helicases appear to be generally oligomeric (usually dimers or hexamers), which provides the helicase with multiple DNA binding sites. The apparent mechanistic similarities and differences among these DNA helicases are discussed.

FtsZ is the major cytoskeletal protein operating in bacterial cell division. FtsZ assembles into protofilaments in vitro, and there has been some controversy over whether the assembly is isodesmic or cooperative. Assembly has been assayed previously by sedimentation and light scattering. However, these techniques will under-report small polymers. We have now produced a mutant of Escherichia coli FtsZ, L68W, which gives a 250% increase in tryptophan fluorescence upon polymerization. This provides a real-time assay of polymer that is directly proportional to the concentration of subunit interfaces. FtsZ-L68W is functional for cell division, and should therefore be a valid model for studying the thermodynamics and kinetics of FtsZ assembly. We assayed assembly at pH 7.7 and pH 6.5, in 2.5 mM EDTA. EDTA blocks GTP hydrolysis and should give an assembly reaction that is not complicated by the irreversible hydrolysis step. Assembly kinetics was determined with a stopped-flow device for a range of FtsZ concentrations. When assembly was initiated by adding 0.2 mM GTP, fluorescence increase showed a lag, followed by nucleation, elongation, and a plateau. The assembly curves were fit to a cooperative mechanism that included a monomer activation step, a weak dimer nucleus, and elongation. Fragmentation was absent in the model, another characteristic of cooperative assembly. We are left with an enigma: how can the FtsZ protofilament, which appears to be one-subunit thick, assemble with apparent cooperativity?

In addition to their mismatch recognition activities, bacterial and eukaryotic MutS activities have an associated ATPase activity that is required for function of the proteins in mismatch repair (1-5). Two distinct functions have been proposed for nucleotide binding and hydrolysis by MutS homologs, both of which are based on the effects of ATP on MutS-heteroduplex interaction. The presence of ATP greatly reduces the efficiency of specific complex formation between bacterial MutS or eukaryotic MutS␣ and heteroduplex DNA (5-10), and ATP challenge of preformed MutS⅐heteroduplex complexes has been shown to result in departure of the protein from the mismatch (11). Available information indicates that some of these effects are attributable to ATP binding. Thus, ATP␥S has been shown to promote departure of MutS from the mismatch in heteroduplex DNA (11), while ATP␥S or ATP (in the absence of a divalent cation) reduce the binding efficiency human MutS␣ (hMutS␣) to synthetic heteroduplexes (5, 10).Electron microscopy of complexes between bacterial MutS and large heteroduplexes prepared from natural DNAs has demonstrated that ATP-promoted release of MutS from a mismatch is associated with efficient conversion of protein⅐DNA complexes to ␣-shaped loop structures stabilized by MutS at the base (11). Loop formation requires a mismatch, loop size increases linearly with time, loop growth depends on continued ATP hydrolysis, and the mismatch usually ends up in the loop. These observations have been interpreted in terms of a mechanism in which ATP binding reduces affinity of the protein for a mispair and activates secondary DNA binding sites that are subsequently used for movement of the protein along the helix contour in a reaction dependent on nucleotide hydrolysis (11). MutS movement in this manner has been postulated to be important for the coupling of mismatch recognition to loading of the excision system at the strand break that directs repair (12, 13), a site that can be located hundreds of base pairs from this mismatch.The finding that ATP binding reduces the efficiency of specific complex formation between hMutS␣ and oligonucleotide heteroduplexes has led to proposal of a molecular switch model for action of MutS activities. Like a G-protein, hMutS␣ is postulated to exist in two states, an ADP-bound form that binds with near irreversible affinity to a mismatch and an ATPbound form that does not (10). In this proposal hMutS␣⅐ADP binds to a mispair and recruits downstream activities to this site. After assembly of the excision system, ATP binding results in dissociation of hMutS␣ from the heteroduplex so that repair may proceed (10).To further clarify the role(s) of ATP binding and hydrolysis in hMutS␣ action, we have evaluated the effects of ATP, ADP, and nonhydrolyzable ATP analogs on the lifetime of hMutS␣⅐DNA complexes and have examined the effect of DNA topology on ATP-promoted dissociation of hMutS␣ complexes with small heteroduplexes. We demonstrate that ADP is not required for mismatch recognition by hMutS␣, but...

Large-scale population based analyses coupled with advances in technology have demonstrated that the human genome is more diverse than originally thought. To date, this diversity has largely been uncovered using short read whole genome sequencing. However, standard short-read approaches, used primarily due to accuracy, throughput and costs, fail to give a complete picture of a genome. They struggle to identify large, balanced structural events, cannot access repetitive regions of the genome and fail to resolve the human genome into its two haplotypes. Here we describe an approach that retains long range information while harnessing the advantages of short reads. Starting from only~ ng of DNA, we produce barcoded short read libraries. The use of novel informatic approaches allows for the barcoded short reads to be associated with the long molecules of origin producing a novel datatype known as 'Linked-Reads'. This approach allows for simultaneous detection of small and large variants from a single Linked-Read library. We have previously demonstrated the utility of whole genome Linked-Reads (lrWGS) for performing diploid, de novo assembly of individual genomes (Weisenfeld et al. ). In this manuscript, weshow the advantages of Linked-Reads over standard short read approaches for reference based analysis. We demonstrate the ability of Linked-Reads to reconstruct megabase scale haplotypes and to recover parts of the genome that are typically inaccessible to short reads, including phenotypically important genes such as STRC, SMN and SMN . We demonstrate the ability of both lrWGS and Linked-Read Whole Exome Sequencing (lrWES) to identify complex structural variations, including balanced events, single exon deletions, and single exon duplications. The data presented here show that Linked-Reads provide a scalable approach for comprehensive genome analysis that is not possible using short reads alone.

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Escherichia coli MutS protein, which is required for mismatch repair, has a slow ATPase activity that obeys Michalelis-Menten kinetics. At 37 degrees C, the steady-state turnover rate for ATP hydrolysis is 1.0 +/- 0.3 min(-1) per monomer equivalent with a K(m) of 33 +/- 6 microM. Hydrolysis is competitively inhibited by the ATP analogues AMPPNP and ATPgammaS, with K(i) values of 4 microM in both cases, and by ADP with a K(i) of 40 microM. The rate of ATP hydrolysis is stimulated 2-5-fold by short hetero- and homoduplex DNAs. The concentration of DNA cofactor that yields half-maximal stimulation is lowest for oligodeoxynucleotide duplexes that contain a mismatched base pair. Pre-steady-state chemical quench analysis has demonstrated a substoichiometric initial burst of ADP formation by free MutS that is governed by a rate constant of 78 min(-1), indicating that the rate-limiting step for the steady-state reaction occurs after hydrolysis. Prebinding of MutS to homoduplex DNA does not alter the burst kinetics or amplitude but only increases the steady-state rate. In contrast, binding of the protein to heteroduplex DNA abolishes the burst of ADP formation, indicating that the rate-limiting step now occurs before hydrolysis. Gel filtration analysis indicates that the MutS dimer assembles into higher order oligomers in a concentration-dependent manner, and that ATP binding shifts this equilibrium to favor assembly. These results, together with kinetic findings, indicate nonequivalence of subunits within a MutS oligomer with respect to ATP hydrolysis and DNA binding.