Download Crystal Structure of a DNA dependent RNA polymerase (DNA

Crystal Structure of a DNA dependent RNA polymerase (DNA
primase)
M. A. Augustin, R. Huber and J. T. Kaiser
Max-Planck-Institut für Biochemie, Abt. Strukturforschung, Am Klopferspitz 18A, D-82152 Martinsried, Germany
DNA primases initiate DNA replication by synthesizing short RNA primers on single-stranded DNA which
are extended by the replicative DNA polymerase(s). In all organisms, primase activity is located on one
polypeptide, however there are considerable variations in the association of primases with other
components of the replication apparatus. In bacteria, the primase complexes with the replicative helicase,
whereas in eukaryotes, the catalytic subunit of primase interacts with DNA polymerase α to form the
tetrameric DNA polymerase α – primase complex. Archaeal primases share a number of conserved motifs
with the eukaryotic enzyme, but an interaction partner has still to be elucidated. Additionally, although
there is a wealth of biochemical data on DNA primases of various sources, structural information is
scarce[1].
We have solved the structure of the primase from the hyperthermophilic archaeon Pyrococcus furiosus
(Pfu-prim) at 2.3 Å by MAD methods using selenomethionine substituted protein. During an initial
absorption scan at beamline BW6, we detected zinc absorption in the crystal, although no zinc was added
during the purification and crystallization procedure. Zinc was found in all well characterized primases, but
due to an unusual binding motif its presence in the archaeal enzyme was questioned[2].
Pfu-prim crystallized in the orthorhombic spacegroup P21212 and is folded into two globular and tightly
packed domains (Fig. 1). The smaller and completely helical domain (top) is highly variable in length and
sequence even within archaea. Its function is unclear, but it might mediate the interaction with accessory
proteins. The larger domain contains most of the residues conserved within eukaryotes and archaea
including the proposed active site which is located in β6 and the linker between α11 and α12.
Figure 1: Stereo ribbon plot of Pyrococcus furiosus primase
It is built up by three aspartic acids which are in a similar three-dimensional arrangement as the `catalytic
triad` residues of DNA polymerases. This finding was very surprising as the overall fold of Pfu-primase
differs extremely from the common fold of DNA polymerases.
In close proximity to the active site, the zinc ion is located in the loop region between β6 and α4. It is
coordinated by three cysteines and one histidine and resembles a zinc-knuckle rather than the well known
zinc finger of other primases. Beginning at the zinc-knuckle, a positively charged cleft runs across the
molecule. It is very likely that DNA might bind in this cleft, as for other primases the involvement of the
zinc motif in binding of single-stranded DNA has been shown. This model suggests RNA synthesis from
right to left using the orientation of Fig. 1. However, this assumption needs to be confirmed by analyzing a
complex of primase bound to single-stranded DNA which we are currently working on.
The results of this study prove that bacterial and archaeal primases show no structural relationship,
suggesting a different evolutionary origin. Furthermore, this structure can serve as a model for eukaryotic
primases and represents an important piece in the puzzle of understanding the replication machinery.
References
[1] J. Keck et al., Science 287, 2482-2486 (2000)
[2] G. Desogus et al., Nucleic Acids Res. 27, 4444-4450 (1999)