Phage Integrase:

Image:  picture of Lambda Integrase, a common phage integrase in the Tyrosine family.^[1]

Outline:         

 1.) What is it?

 2.)How it works?

 3.)Why is it important?

 4.)Structure?

What is it?

Bacteriophage are viruses that infect host bacteria.  These viruses are responsible for a major pathway as to how bacteria exchange genetic material.  These bacteriophages prove to extremely important in modern molecular biology. (www.phage.org)  Phage integrases are enzymes that mediate unidirectional site specific recombination between two DNA recognition sequences, attP (phage attachment site) and attB (bacterial attachment site). (www.ncbi.nlm)  The minimal sizes for attB and attP  are 34 bp and 39 bp, respectively (Groth et al, 1999).  The integrase protein is produced by a gene that is turned on by gene N in the phage DNA.^[2]  Phage integrase is made up of three domains which include arn-type binding domain, core-type binding domain, and the catalytic domain. (Cho et al, 1999)  These proteins are very specific in the sense that they only recognize their specific attachment sites but not those of other recombinases due to evolutionary diversity.^[3]  They can be divided up into two major groups which include: Tyrosine recombinases and Serine recombinases.  Tyrosine integrases utilize a catalytic tyrosine to mediate strand cleavage while the Serine integrases use a catalytic serine to mediate strand cleavage.  Tyrosine family was classified by 27 proteins but has now raised to 81 proteins.  Previously, this family shared four strongly conserved residues but now more have been identified (Esposito et al, 1997).  The Serine family were thought to be highly regulated and only be responsible for resolution or inversion.  However recent studies show that the Serine family is very diverse such that transposases can be included which are responsible for integration and excision (Smith et al, 2002). ^[4]

                                        Tyrosine Recombinases                                Serine Recombinases

                                       -recognizes longer attP sequences                 -recognizes shorter attP                                                                                                                              sequences-

                                       -require other proteins encoded by the             -no required host cofactors 

                                        phage or host bacteria                                  -larger in size

                                       - Ex: -lambda integrase                                 -known as resolvase/invertase           

                                               -Cre recombinase                                  -Ex: -gamma/delta resolvase

                                                                                                                  -Tn3

How it Works? 

The mechanism behind this group of enzymes: (Alberts et al, 2007)

They carry out recombination by covalently attaching the DNA transiently to the enzyme.  Two integrase monomers are bound at each attachment site (att).  The hydroxyl group of the Tyrosine nucleophilically attacks the phosphate backbone of DNA causing it to break.  For each att site, the 3' end of one DNA strand is covalently bound to an integrase protomer which leaves a free 5' OH.  This free 5' OH attacks the protein-DNA bound to the opposite att site resulting in the formation of a holliday junction.  A small branch migration occurs and the process repeats to resolve the junction.  For the Serine integrase, the hydroxyl group of the Serine nucleophilically attacks the phosphate backbone of DNA.  It attaches to the 5' DNA ends leaving free 3' OH groups.  The Serine family makes a 2bp staggered cut at each att site, breaking all four stands at once. This is followed by a 180 degree rotation and DNA ligation.  An image of this mechanism which phage integrase uses to insert phage DNA into bacterial DNA sequence can be seen. (UC Berkeley project)  There is a animation of how integrase works in site-specific recombination.( www.Harvard.com) ^[5] 

Why is it Important?

The properties of phage integrases make them increasingly important in the genetic manipulation of living eukaryotic cell, especially those with large genomes such as mammals and plants.  Specifically integrases of the Serine family have been shown to be useful in mammalian cells, mediating efficient integration at introduced att sites and native sequences.  Some of these Serine family integrases that are used include: Cre, Flp, and phi C31. (Wirth et al, 2007) These integrases are allowing scientists to conduct much more accurate and cheaper research because they don't have to rely on general recombination; site specific recombinases don't require tedious labor and screening as general recombination does.  They are very useful in the field of gene therapy, construction of transgenic organisms, and manipulation of cell lines.  Directed evolution may also open up possibilities for further genomic modifications.  They have become important tools for developmental and cell biologists.  Using carefully designed target DNA with DNA sites the enzyme recognizes, they can decipher the roles of specific genes and proteins in complex organisms such as mice.  They can delete specific genes in specific tissues of the mouse.  This becomes very useful when the gene plays an important role in the early development of many tissues.  The integrase can also be used to inappropriately express a gene in a tissue of interest.  Together the researchers becomes able to determine the role of any protein in any desired tissue of an intact animal.  Custom integrases can be created which can be applied to large number of applications.  These custom integrases can be increased in specificity and efficiency in human cells.  ^[6]

Integrase can also play an integral role in treating a disease.  HIV I disease is an immunosuppressive disease that is caused by a retrovirus.  This virus uses integrase to successfully replicate.  Integrase (INT) serves as a target for antivirus drugs due to their highly conserved nature on the pol gene along with other regions. (www.NewYorkTimes.com, 2007) An integrase inhibitor has been formulated in order to treat the HIV I by not allowing the virus to replicate. (Dessalew, 2009) ^[7]

Attacking the integrase protein is a good target for treating HIV because it isnt used by humans to perform necessary function so a integrase inhibitor won't harm the normal function of human cells. (www.aidsmed.com)

Integrase Problems:

Integrase is a very important and diverse group of proteins.  They are for the site-specific recombination that occurs in both prokaryotes and eukaryotes.  However, problems can arise in the integrase proteins which cause many problems.  An example of a negative effect of integrase can be seen in human fibroblasts which when express a specific integrase leads to DNA damage and chromosomal problems. (Liu et al, 2009)  Also, without proper function of integrase, phage wouldn't be able to integrate its DNA into that of bacteria and different eukaryotes (research).  This would cause a decrease in bacterial diversity and eukaryotic gene research.