Macromolecular Structures Group - Projects
Protein-DNA Interactions
(M. Kokkinidis, A. Athanasiadis, D. Kotsifaki, M.
Papadovasilaki, Y. Papanikolaou, K. Petratos, M. Vlassi)
In this field we are pursuing
the study of three types of interactions, i.e., those of type II restriction endonucleases,
DNA-methylases and zinc fingers. The work on the former two types is mainly
crystallographic, while the zinc-fingers interactions are being approached via a
statistical analysis of their sequences and their relation to their DNA targets.
2.1 Type II Restriction Endonucleases
These enzymes cleave double-stranded DNA in the
presence of Mg2+ ions with extremely high specificity at fixed locations within or near
defined recognition (cognate) sequences. Nearly 2000 type-II restriction endonucleases
have been identified but only very few have been studied until recently in detail, i.e., in
the crystallographic determination of their 3D-structures. Despite their common function,
these enzymes constitute at the level of amino-acid sequences a diverse set of proteins.
A major step towards a systematic approach of structure-function relationships in type II
endonucleases was the recent determination at 2.5 ngstroem resolution of the 3D-
structure of the dimeric Pvu II restriction endonuclease (Pvu IIR) by our group. Despite
a nearly complete lack of sequence homology to other endonucleases, the protein
shows a striking similarity in its major topological elements to EcoR V. On the basis of
this observation we have been able to propose a structural classification of the type II
restriction endonucleases. Furthermore, we have proposed a model for the early event
occurring during DNA recognition. Further open questions, e.g. the role of structural
flexibility, the basis of the star activity etc., are presently being investigated. Co
crystals of the enzyme with an oligonucleotide containing the sequence associated with
the star activity of the protein have recently been obtained.
2.2 Methylases
In contrast to
type II restriction endonucleases, methylases show considerably more sequence
similarities and share a considerable number of sequence motifs. Depending on which
atom the methyl group is transferred to, methylases are classified as m5C-methylases
(transfer to the C5 atom of cytosine bases), m4C- (to the N4 atom of cytosines) and
m6A-methylases (transfer to the N6 atom of adenines). The limited structural
information available on methylases makes the recent crystallization of the m6A
methylase BseC I (BseC IM) by our group an interesting crystallographic project. BseC
IM from Bacillus stearothermophilus, an isoschizomer of Cla I, is a thermostable
enzyme of 578 amino acids with a molecular weight of 66.7 kDa. The enzyme has been
crystallized using PEG 6000 in space group P21 with a=53.7, b=85.7, c=151.8
(Angstroem) and â=95.1o and two molecules in the asymmetric unit. The diffraction limit
of the crystals is better than 2.5 Angstroem. Data sets collected at 100 K and at room
temperature clearly show the non-crystallographic twofold axis. We have now initiated
the search for isomorphous heavy atom derivatives.
2.3 Zinc-fingers
A more theoretical
and (bio)computational approach is pursued in the study of zinc-finger sequence-
topology relationships. The work was initiated with the modelling of the complex
between three fingers of the CF2 protein and their cognate DNA sites (in collaboration
with F.C. Kafatos and J. Gogos). The interactions of a large number of site-directed
mutants of the CF2 protein with DNA confirm our model, which is not a simple extension
of PaboÕs first crystallographic structure but allows us to make several predictions
concerning the topology of the interaction, the role of structural flexibility etc. We have
now generalized these concepts in a statistical approach for the structure-sequence
relationships of zinc fingers.
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