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Prokaryotic Replicative Helicase Models

Helicases are ubiquitous and integral members of almost all complexes that catalyze reactions of nucleic acid metabolism (Patel & Picha, 2000; Lohman & Bjornson, 1996; Matson & Kaiser-Rogers, 1990 ). They are motor proteins that couple the hydrolysis of nucleoside triphosphate (NTPase) to nucleic acid unwinding. The DNA helicases involved in replication, repair and recombination work in association with other proteins as part of a complex machine (Kornberg & Baker, 1992).Most helicases, however, are able to catalyze by themselves the strand separation when a suitable substrate is provided in vitro. The exact mechanism by which helicases accomplish this reaction remains unclear.

Prokaryotic DNA replicative helicases are homohexamers of a characteristic ring-shaped structure. All known helicases have been grouped into families and superfamilies based on their primary amino acid sequences (Gorbalenya & Koonin, 1993; Ilyna et al., 1992).The hexameric helicases do not fall within a single family, but are members of various families.Prokaryotic DNA replicative helicases belong to the DnaB-like family, whose flagship member is DnaB of E.coli.Members of this family of enzymes have five conserved motifs (H1, H1a, H2, H3, H4) in the helicase domain.The H1 and H2 motifs contain the Walker A and B sequences (Walker et al., 1982).The crystal structures of two prokaryotic hexameric helicases are known, those of gp4 of bacteriophage T7 (Singleton et al., 2000; Sawaya et al., 1999) and RepA of plasmid RSF1010 (Niedenzu et al., 2001).As expected, both proteins possessed the so-called RecA fold and closely resembled the F1-ATPase monomer structure.

Quaternary polymorphism is a distinctive structural feature of members of the DnaB-like family of replicative helicase proteins.Such phenomenon consists in the existence of hexamers with architectures of different rotational symmetry, mainly 3-fold (that can be pure C3 or mixed C3C6) and 6-fold (C6). It was first observed, by electron microscopy, in DnaB (Yu et al., 1996) and later described, also by the same technique, in the replicative helicases RepA of plasmid RSF1010 (Bárcena, 2000) and G40P of bacteriophage SPP1 (Bárcena et al., 1998) Although it is not known the precise role of quaternary polymorphism, this capability must be significant in the functioning of these enzymes, either for their helicase activity, loading onto the replication fork or interaction with other proteins of the replication machinery.

Our group is devoted to the structural analysis of a range of DNA helicases by means of transmission electron microscopy, of both negatively stained and frozen-hydrated samples, and digital image processing and three-dimensional reconstruction techniques.

Cryo<-EM structure of DnaB and DnaB·DnaC

E.coli has at least 12 different enzymes that act as helicases. Among them, DnaB is the main replicative helicase, the factor primarily responsible for the unwinding of the DNA duplex at the replication forks in chromosomal DNA synthesis.

In solution, the hexameric DnaB forms a complex with six molecules of DnaC.  DnaC is essential for replication in vitro and in vivo. The role of this protein centres around formation of the DnaB·DnaC complex, from which it delivers the helicase to its site of action on the DNA template.

Following up previous studies performed on negatively stained samples (San Martín et al., 1995), our group studied the structure of both the DnaB hexamer and the DnaB·DnaC complex by single-particle 3-D reconstruction from cryo-electron microscopy images of frozen-hydrated specimens, in collaboration with the group of Prof. N.E. Dixon (Australian National University, Canberra). Those were the first reports of the 3-D structure of a helicase obtained by this technique, that also provided the first insights into the structure of a complex of a helicase with its loading protein (San Martín et al., 1998; Bárcena et al., 2001).

The DnaB oligomers present six density maxima arranged in a triangle-shaped particle. The edge of the particle is 10-11 nm long, the external diameter of the particle is 12.5 nm, and the reconstructed height is 5.7 nm. The density lobes are organized in a ring around a channel running open from face to face of the hexamer. The channel has a diameter of about 3 nm. The six subunits in the hexamer do not have the same aspect, as three of them seem significantly larger that the others. Subunits with each of the two aspects are positioned alternately around the ring. The two faces of the hexamer are different, with a clear transition from six-fold to three-fold symmetries between them.

Surface rendering of the reconstructed volumes of DnaB (left panel) and DnaB·DnaC complex (right panel) at low (chicken wire) and high (solid yellow) threshold values, to emphasize the highest density areas and the contacts between them.

The 26 Å resolution three-dimensional structure of the DnaB·DnaC complex obtained from cryo-electron microscopy revealed the elaborate way in which the DnaB and DnaC proteins interact within the complex and provided a structural basis for control of the symmetry state and inactivation of the helicase DnaB by its loader DnaC.The complex is arranged on the basis of interactions between DnaC and DnaB dimers. DnaC were observed foe the first time to arrange as three dumb-bell-shaped dimers that interlock into one of the faces of the helicase. This could be responsible for the freezing of DnaB in a C3 architecture by its loading partner. The central channel of the helicase is almost occluded near the end opposite to DnaC, such that even single-stranded DNA could not pass through.

Quaternary polymorphism in the helicases DnaB and G40P.

We first described, in collaboration with the group of Prof. J.C. Alonso (C.N.B.), the polymorphic quaternary organization of the B.subtilis bacteriophage SPP1 helicase G40P (Bárcena et al., 1998).Our study provided the first low resolution data on a hexameric helicase of a Gram-positive bacterial origin. A prototype of a novel approach, later on improved also in our group (Pascual-Marquí et al., 2001; Pascual-Montano et al., 2001), was used to analyze possible symmetry heterogeneities, an unsupervised method based on a neural network self-organizing algorithm, that led to the detection of different subclasses of G40P views.Two different quaternary states of G40P hexamers sharing a C3 symmetry organization were found together with a third class, of C6 symmetry architecture.  All forms showed general features known for other hexameric helicases.A clear structural handness was also detected on some of those forms.

Quaternary polymorphism of helicase G40P.Average images for the three main classes detected (top panel), their corresponding rotational power spectra (middle panel) and contour maps of the symmetryzed average images.

In a different work, electron microscopy and in-depth rotational analysis studies of negatively stained specimens allowed us to establish certain experimental conditions that govern the transition between the two different rotational symmetry states (C3 and C6) of DnaB in vitro (Donate et al., 2000). It was shown: (a) that the pH value of the sample buffer, within the physiological range, dictates the quaternary organization of the DnaB oligomer; (b) that the pH-induced transition is fully reversible; (c) that the type of adenine nucleotide complexed to DnaB, whether hydrolysable or not, does not affect its quaternary architecture; (d) that the DnaB·DnaC complex exists only as particles with C3 symmetry; and (e) that DnaC interacts only with DnaB particles that have C3 symmetry.

References

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