Genetic Recombination in Bacteria PDF

Genetic Recombination What happens to foreign DNA in cell? A. Degraded B. Replicates independently of the chromosome Plasmids and some viruses C. Inserts into host DNA  Recombination Two molecules of DNA with nearly identical sequences can recombine Genetic Reco...

Genetic Recombination What happens to foreign DNA in cell? A. Degraded B. Replicates independently of the chromosome Plasmids and some viruses C. Inserts into host DNA  Recombination Two molecules of DNA with nearly identical sequences can recombine Genetic Recombination: Within living cells, the exchange of DNA sequences and genetic information occur through an intricately regulated series of enzymatic reactions involving pairing of DNA molecules and phosphodiester bond breakage and rejoining. 1. Homologous recombination 2. site specific recombination 3. Illegitimate recombination  Homologous recombination Substrates The common stretch of bases that will be recombined is known as homology Proteins 1. Identical or similar seq 2. Ability to form H bonds 3. Proteins needed (endo/exo nuclease, RecA, ligase) 50-100 bp to even whole Products genome Chi (crossover hotspot instigator) About 1000 chi sites in E.coli RecBCD complex (330 kDa) Exonuclease V, helicase, ATPase 10 to 50 RecBCD molecules per cell One RecA binds per 5 bp of DNA It is DNA dependent ATPase RecA-ssDNA filament binds to another strand and unwind by breaking hydrogen bonds; further promoting recombination with another DNA  Earlier models of recombination Chaismatype theory John Belling (1931) Janssens (1909) and Darliington (1930) Alfred Sturtevant (1949) Joshua Lederberg (1955)  Harold Whitehouse and Robin Holliday Holliday Model (1964) Breakage Disassociation and assimilation (RecA) Ligation Branch migration (RuvABC) RuvA and RuvB -branch migration proteins, RuvC -junction-resolving enzyme (nuclease) Isomerization & Resolution ligation Single strand invasion Model Meselson and Radding (1975) Site specific recombination Illegitmate recombination In absence of RecA, regions of DNA homology or specific sites Spontaneous DNA arrangements such as deletions, duplications Associated with very small repeated sequences Short sequence repeats (SSR) Mechanisms of Gene transfer among Bacteria Transduction Zinder and Lederberg, 1952 Lysogenic cycle Generalized Transduction  Specialized Transduction  Transformation Recombination or replication of DNA is required for stable maintenance of DNA  Do all cells take up naked DNA? No Cell can be made competent to take up DNA Natural competence Chemically induced competence Electroporation is another way to force uptake of naked DNA Natural Competency Competency is induced when – Number of cells in a given volume increases or – Shifting of cells from optimal growth conditions to poor growth conditions – Pathway 1-more no. of cells – Pathway 2-monitors the depletion of available nutrients – Signals the cells to induce com genes Com Q protein processes and modified precursor comX to make smaller active peptide. The active comX is excreted from the cell Quorum sensing system Autophosphorylation in histidine of sensor kinase Phosphate transferred by sensor kinase to an aspartate in the response regulator protein The phosphorylated RRP is able to either turn on or off the genes transformation  Conjugation Requires donor and recipient cells Donors must contain a conjugative plasmid encodes genes for conjugation One conjugative E. coli plasmid is called the F plasmid Genetic Map of the F plasmid RepF1A – OriS, OriV RepF1B – backup OriT Relaxase enzyme (TraI) Relaxasome (TraH, TraL, TraJ, TraY, TraK and TraM) + IHF TraS and TraT RepF1A – OriS, OriV RepF1B – backup Relaxasome (TraH, TraL, TraJ, TraY, TraK and TraM) TraI - Relaxase enzyme TraM –helps in relaxase interaction TraH, stabilizes the relaxosome's structural formation TraJ – recruits the complex to oriT site, TraK – nicked state; TraY – ssDNA state stability; IHF; TraS and TraT Relaxosome: – Nicking: – Unwinding: – Attaching:TraI to the 5′ end of the nick – Transporting Surface exclusion – Greatly reduces transfer of multiple F factors into the same cell – TraT inhibits mating pair formation – TraS-inhibits DNA transfer The F+ cell donates to an F- cell. Rolling circle replication is used. F plasmid can replicate independently of the chromosome or it can integrate into the chromosme Map of a drug resistance plasmid