Holliday Model for Homologous Recombination

• Two DNA molecules with nicks induced by an endonuclease in strands of the same polarity can invade each other with free single strands.
• A Holliday junction, or Chi structure, is formed as a recombination intermediate, and is solidified by ligase sealing the nicks, uniting the two homologues.
• Branch migration allows exchange of material, since the loose area around the junction allows unzipping/rezipping.
• New nicks allow separation of recombined DNA and resolution of the recombination intermediate.
• The Holliday model assumes reciprocal and equal exchange of genetic material between DNA molecules.

Fig. 31. Holliday model

Double-Strand Break Repair Model (Szostak Model) for Recombination

• Double-strand breaks are introduced into one of the homologues.
• Broken ends are recessed by specific exonucleases which produce longer 3' single strands.
• The single strands can invade a region of homologous DNA.
• The invading end serves as a primer for a polymerase which extends it, unwidnding the template DNA and generating a D-loop. A Holliday junction is formed, and DNA synthesis proceeds until it reaches the opposing end of the recombining chromosome.
• The displaced DNA of the opposing end of the invading chromosome anneals to the template (invaded chromosome), forming a second Holliday junction by ligation of the strands. Holliday junctions can migrate prior to resolution with resolvase endonucleases.
• Resolution can cut the invading strands, resulting in a non-crossover event, since each chromosome has only a tiny fragment of DNA from the other chromosome.
• Crossover results from cutting the strands not participating in the recombination, generating a crossover by leaving a large segment of each chromosome attached to the other.
• The Szostak model accounts for non-reciprocal recombination.

 

Fig. 32. Double and single strand break repair model

Factors Involved in Each Recombination Step

• The Szostak model is an error-free pathway (since it can only occur in homologues), and is of major importance in yeasts and mammals.
• A 5' to 3' recession reaction is mediated by endonucleases.
• Strand invasion requires multiple Rad proteins, including DNA helicases and DNA end-binding proteins.
• New DNA synthesis requires DNA Pol δ and ε (delta and epsilon).

Repair of DSBs by Non-Homologous End Joining (NHEJ)

• NHEJ is an error-prone pathway, and is the major pathway in mammals for repair of DSBs.
• DNA-PKcs (DNA protein kinase, catalytic subunit) binds ends of the DSB (double-strand break).
• Endonucleases create blunt (no overhang) ends.
• Synapsis is achieved through microhomologies.
• Ends are ligated, and, due to end-blunting and other factors, small insertions and deletions are introduced. The errors are often of little consequence, since most DNA is non-coding.

Site specific recombination

Site-specific recombination , also known as   conservative site-specific recombination , is a type of   genetic recombination   in which   DNA   strand exchange takes place between segments possessing only a limited degree of   sequence homology.   Site-specific   recombinases   perform rearrangements of DNA segments by recognizing and binding to short DNA sequences (sites), at which they cleave the DNA backbone, exchange the two DNA helices involved and rejoin the DNA strands. While in some site-specific recombination systems just a   recombinase enzyme   and the recombination sites is enough to perform all these reactions, in other systems a number of accessory proteins and/or accessory sites are also needed.

Site-specific recombination systems are highly specific, fast and efficient, even when faced with complex   eukaryotic   genomes.   They are employed in a variety of cellular processes, including bacterial genome replication, differentiation and pathogenesis, and movement of   mobile genetic elements. For the same reasons, they present a potential basis for the development of   genetic engineering   tools.

Recombination sites are typically between 30 and 200   nucleotides   in length and consist of two motifs with a partial inverted-repeat symmetry, to which the recombinase binds, and which flank a central crossover sequence at which the recombination takes place. The pairs of sites between which the recombination occurs are usually identical, but there are exceptions (e.g. attP and attB of   λ integrase,)

Fig. 33.   Insertion and excision mediated by aligned Lox sites and the cre recombinase. Red X designates recombination.  

 

REFERENCES:

Text Books:

1. Jeffery C. Pommerville. Alcamo's Fundamentals of Microbiology (Tenth Edition). Jones and Bartlett Student edition.

2. Gerard J. Tortora, Berdell R. Funke, Christine L. Case. Pearson - Microbiology: An Introduction. Benjamin Cummings.

3. J. Krebs, E.S. Goldstein, Stephen T. Kilpatrick. Lewin's Genes X. Jones and Bartlett Publishers.

Reference Books:

1. Lansing M. Prescott, John P. Harley and Donald A. Klein. Microbiology. Mc Graw Hill companies.

Other References:

1. http://www.nature.com/scitable/topicpage/genetic-recombination-514