HOMOLOGOUS AND SITESPECIFIC RECOMBINATION Dr Nket Yrr Kutlay
HOMOLOGOUS AND SITE-SPECIFIC RECOMBINATION Dr. Nüket Yürür Kutlay
HOMOLOGOUS RECOMBINATION Homologous (general) recombination; genetic exchange takes place between a pair of homologous DNA sequence ( similar or identical nucleotide sequence) The fundamental processes that catalyze homologous recombination are common to all cells (bacteria, viruses, yeasts) The total number of nucleotides is not changed.
Homologous recombination is used; • to repair DNA (the most widespread use is in accurately repairing double-strand breaks) • to exchange bits of genetic information between two different chromosomes to create new combination – Gene conversion – Crossing-over in meiosis • to assure an accurate chromosomal segregation. (by playing an important mechanical role in meiosis)
Repair of a single strand breakage During the replication, ehead of a replication fork Figure 5 -53 Molecular Biology of the Cell (© Garland Science 2008)
One broken and one intact daugher chromosome The initial strand invasion requires a free 3’ end generated by nuclease that degrades the 5’ end of the complementary strand
• It takes place only between DNA duplexes that have extensive regions of sequence similarity ( homology ) • Base-pairing is a requirement for a perfect match and successful hom. rec.
Base-pairing interaction can be mimecked in a test tube by allowing a DNA double helix re-formed; Random collision complementary base pairs have formed rapid zippering formation of complete double helix
• Proteins have an important role in the basepairing (to make it easier) • Renaturation / hybridization • Base pairing can not be done between two intact DNA double helices – strand invasion – Rec. A is bound to a DNA single strand
• For an unfolded conformation of annealing strands, SSB proteins are used (tightly and cooperatively bind to sugar-phosphate backbone) – DNA replication – Homologous recombination • Heteroduplex is an essential step in any hom. rec. process. “a region of DNA helix formed from strands that originate from two different DNA molecules.
Rad 51(human homolog of bacterial Rec. A ) helical filament
Rec. A filament can hold a single strand double helix together, and catalyze a multistep DNA synapsis. Figure 5 -56 Molecular Biology of the Cell (© Garland Science 2008)
in addition to rec. A(Rad 51); - Rad 52 displaces SSB protein, allowing the binding of Rad 51 -
After heterodublex region is formed, it is often enlarged by a process called branch migration By specialized DNA helicases Unidirectional Figure 5 -58 Molecular Biology of the Cell (© Garland Science 2008)
Repair of a double-strand breakage Shortly after the DNA has been replicated and two sister chromatids are still held together. Daughter DNA duplex can serve as the template for repair of the other Without any loss or alteration of nucleotides at site of repair. Figure 5 -59 Molecular Biology of the Cell (© Garland Science 2008)
During hom. rec. a special DNA intermediate forms; Holliday junction(cross-strand exchange). It contains four DNA strands shared between two DNA helices Transiently present Figure 5 -61 Molecular Biology of the Cell (© Garland Science 2008)
Ruv A(green) tetramer Ruv B(g) Hexamer helikaz (uses energy of ATP hydrolysis) This junction must be cut for seperation; a process refered to as resolution. Ruv C endonuclease Ruv. C (SLX 1/SLX 4, GEN 1) or Rec. Q (BLM and TOPIIA) Figure 5 -62 Molecular Biology of the Cell (© Garland Science 2008)
As a result of meiotic recombination , hybrid chromosomes are produced. Figure 5 -63 Molecular Biology of the Cell (© Garland Science 2008)
Meiotic recombination begins with a programmed double strand break Protruting 3’single strand ends Budding yeast Figure 5 -64 Molecular Biology of the Cell (© Garland Science 2008)
• Meiosis/DS break repair – As well as proteins that function in DS break repair, several specific proteins serve in meiosis – In DS break repair, hom rec occurs between identical DNA duplexes In meiosis, hom rec occurs between Pat-Mat chromosomal homologs
- Few of Spo 11 mediated DS breaks become crossover. The majority are resolved as noncrossover(%90) - This choice is made before the Holliday junction are formed - Crossover control; not closely placed. One crossover on each arm of chromosome occurs during meiosis
Event can be resolved as a crossover or a non-crossover, in both ways, heteroduplex regions remain 1000 bp Small percentage of mismatched base pairs can be tolerated. Heteroduplex regions are potential gene conversion sites Figure 5 -65 Molecular Biology of the Cell (© Garland Science 2008)
Gene conversion: - Corrected by mismatch repair machinery - Strand which will removed is randomly selected
Homologous recombination is carefully regulated • Loss of heterozygosity • Recombination-based repair is prevented in the absence of DNA damage; – BRCA 1, BRCA 2 RAD 51(binding and preventing its polymerization on DNA, thereby maintaining it in an inactive form /DNA damage; bring it and release it) • Too much or too little homologous recombination can lead to cancer
Transposition(al rec) Conservative site-specific recombination • Can alter gene order along • Mobile genetic elements chromosome • Range in size; 100 -10000 bp • Jumping genes/ selfish DNA/ • Cause unusual types of mutations mol. parasites • Had a profound effect on the shaping of modern genome • A few of them in our DNA are still active • Carries a unique set of genes
• Mobile elements that move by transposition are called transposons or transposable elements • They are only modestly selective in choosing their target side • Move rarely
Three Major Classes of Transposoable elements • DNA only transposons • Retroviral like-retrotransposons • Nonretroviral retrotransposons
DNA transposons Once every 105 cell divisions Horizontal gene transfer
DNA only transposons; mode of movement * Cut and paste pathway like VDJ * Replicative pathway
• Life cycle of retrovirus (AIDS/HIV), integrase • Retroviral-like retrotransposons • Unlike viruses, they have no intrinsic ability to leave their resident cell
Nonretroviral retrotransposons • mode of movement; – Moves via an RNA • Reverse transcriptase and endonuclease
• 35% of genome • L 1(LINE): long interspersed nuclear element • SINE: Short interspersed nuclear element(Alu)
Together the LINEs and SINEs make up abouth 40% of human genome Human genome contains all three types of transposon
Transposons and their activation situation varies according to species. • In bacteria; mostly DNA transposons • In yeast; mostly retro viral like retrotransposons • In drozofila; all three transposons
Conservatif site-spesific recombination; requires specialized DNA sequence recombinases form transient high-energy covalent bonds with DNA and use this energy to complete the DNA rearrangements A bakteriofaj /B(Salmonella flagelin)
Alberts • Johnson • Lewis • Raff • Roberts • Walter Molecular Biology of the Cell Fifth Edition Chapter 5 DNA Replication, Repair, and Recombination Homolog Recombination Transposition and Concervative site-specific Recombination Copyright © Garland Science 2008
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