Scientific Programmes
Structural Biology and Biocomputing Programme
Macromolecular Crystallography Group
Overview Last update May/2010
S-phase and replication
DNA replication is an essential process during cell division. The identification of the DNA helicase(s) involved in eukaryotic DNA replication is still a matter of much debate. Recently, the helicase activity of the hexameric MCM complex has been revealed as being responsible for the unwinding of DNA during S phase in association with two partners: initiation factor Cdc45 and a four-subunit complex called GINS. In conjunction they form the CMG complex which contains ATP dependent helicase activity. We aim to unravel the molecular mechanisms of this cellular machinery essential for eukaryotic DNA replication. During this year we have been able to obtain all these components using co-expression techniques. Thus, we are now able to study them by combining X-ray crystallography and EM studies to decipher the structure of this complex and its components.
Mitotic complexes
Cellular growth and division are regulated by an integrated protein network which ensures the genomic integrity of all eukaryotic cells during mitosis. This cell cycle stage witnesses a massive reorganisation of cellular architecture. All these events need the assistance of different proteins to ensure their proper protein folding and performance. One protein that performs this function is a eukaryotic macromolecular complex that allows tubulin, an essential cytoskeletal molecule, to fold properly and form the mitotic spindle among other structures. We have solved the structure of this macromolecular machine, in complex with tubulin, one of its main substrates. Our objective is to obtain high resolution information regarding the atomic structure and the regulation of these molecules.
Structural design of homing endonucleases for gene targeting
Homing endonculeases or meganucleases are sequencespecific enzymes which recognise large (12–45 bp) DNA target sites. These enzymes are often encoded by introns or inteins behaving as mobile genetic elements. They recognise sites that usually correspond to intron-free or intein-free genes, where they produce a DNA double-strand break (DSB). Eventually, DSB repair by homologous recombination with an intron – or intein – containing gene results in the insertion of the intron or intein where DSB occurred in specifi c loci in living cells.
These results present new perspectives in a wide range of applications, such as the correction of mutations linked with monogenic inherited diseases. Our Group has participated in the development of a chimaeric enzyme that could target mutations in the RAG gene promoting its repair. In addition we have shown that repair of the gene can be done in its locus in human cells, opening avenues to possible therapeutic applications. The crystal structure of monomeric meganuclease I-Dmol in complex with its target DNA has allowed us to turn this endonuclease into a nicking enzyme, providing us with a new tool to repair genes preferentially using DSB homologous recombination. DNA nicks are favourably repaired using this route to avoid the unsafe non-homologous end joining (NHEJ) pathway that promotes loss of genetic information.
