The Theory of Binding Sites

In the study and analysis of the characteristic of the gyrase-DNA complex , Morrison and Cozzarelli (1981) had found out that there are several consensus sequence in DNA which favours the binding of gyrase and subsequent catalysis . Their experiments involved uses DNase I and examined the DNA sequences which were protected from the digest by the endonuclease.

These highly preferred or specific sites are know as gyrase binding sites (GBS) or strong gyrase sites and are found to be about 280bp ( Mu phage SGS) according to Oram(2003). In the 280bp DNA sequence, approximately 130bp will be wrapped around the tetrameric enzyme in a way similar to nuclesome formation. The gyrase will them introduced a 4bp staggered breaks in the DNA and alter the DNA linking number ( please refer to Modelling and Supercoilogy theory for explanation) .

In the in vitro pBR322 plasmid GBS study, experts identified that the preferred cleavage sites have the following DNA sequence , 5’-GGCTGGATGGCCTTCCCCAT – 3’ . Through the study in identifying the consensus sequence for gyrase binding, researchers found out that cleavage occurs between the TG residues ( 990th position in pBR322) in the DNA sequence. By multiple sequence alignment and mathematical prediction , it is estimated that there are around 45 to 50 major gyrase binding sites in the E.coli genome. These sites play an important role in compacting the bacteria chromosomal nucleoid as well as regulation of transcription and DNA replication. Apart from the major binding sites , there are about 10 000 more of the weak binding sites , but gyrase will not normally bind to these sequences unless they are in an extremely high concentration.

Apparently some bacteriophages also exploited the GBS to allow them to replicate successfully in their host. For example, Mu – bacteriophage has a strong gyrase site or GBS in the central region of the 37.2kbp Mu phage genome. The ‘essential’ 147bp sequence in the region is vital to the phage replication within the bacteria host . It is believed that Mu phage DNA replication requires the phage DNA to be negatively supercoiled at specific positions in the genome until a certain extent which enables DNA replication to be efficiently carried out. The introduction of the highly preferred sites allows the phage to ‘hijack’ the endogeneous E.coli gyrase enzymes to supercoil their own DNA.

A PICTURE

The figure above is a study from Pato(1990) which indicate the importance of the essential 147 bp in the Mu-GBS . The wild type phage can carry out replication effectively ( hence higher percentage of the incorporation of radioactive tritium nucleotides) compare to the mutant which lacks the GBS after the heat induction of the lysogens . However the above study is carried out in vitro , in vivo studies will involve more factors and is not clearly understood how the results will deviate from the one obtained in vitro.

Thus, in 2011 UCL iGEM project , we have study the usage of such sites to improve the supercoiling consistency and specificity of the target plasmids . By this method , we hope to achieve better supercoiling without interfering with the normal physiological E.coli chromosomal supercoiling which may in turn affect the growth of the bacteria.