Recombinational Repair• Replication though a single strand breaks collapses the replication fork • Homologous recombination re-establishes replication• Double strand breaks (DSBs) can also be caused by:◦ Oxidative damage ◦ Radiation damage ◦ Cellular nucleases Homologous (nearly identical) Recombination • A DNA rearrangement-DNA is cute and rejoined which can generate new combinations of genes• Important for DNA repair (ds breaks)• Required for meiosis in eukaryotes in eukaryotes (crossover between homologous chromosomes are required for proper chromosone segregation and for generating diversity in the gametes • Allows genes to be transmitted from one bacterial strain to a closley related strain • Takes place in all organisms• Homologous Recombination in E. coli ◦ REC BCD • Involved in recombination events that are promoted by double stranded breaks during conjugation or replication fork collapse • Going to trim back the DNA at the site of the double strandbreak on both sides • Has 5` --> 3' and 3'-->5' exonuclease activity at the end helices • Binds to the double stranded break, unwinds the strand • And degrades each strand independently from one another• 3'--> 5' exonuclease activity is faster so that the 3' end gets degraded faster and leaves a 5' overhang but the strand degradation will proceed until it reaches the chi site (cross over hot spot instigator)• Chi site has a characteristic sequence (5'-GCTGGTGG-3') • At chi site, the exonuclease activity happens ( the 3' to 5' exonuclease activity decreases whereas the 5' to 3' exonuclesae activity increase • Going to leave an overhang 3' overhang to allow strandinvasion • RecBCD-helicase nuclease binds to breal, helicase unwinds and nuclease degrades both strands• When RecBCD encounters a chi site (5-'GCTGGTGG-3')its activity changes ▪ Stops degrading the strand with 3' end and becomes more active degrading the strand with the 5' end▪ This generates the 3' tail needed to initiate recombination• RecA (required for all homologous recombination) mediates strand invasion◦ Its mutants don't do homologous recombination ◦ its conserved throughout evolution (Rad51 --> eukaryotic version)◦ Intially binds to a single strand and forms filament 5' to 3'◦ Then searches for homology and initiates strand invasion and duplex formation ◦ Requires at least 50 bp of homology (the longer the better)• RecA is going to load on the 3'end --> going to facilitate invasion into homologous duplex • Then we have strand invasion, formation of the D-loop , DNA will through recombination◦ RuvABC will act on the holiday junction• We need cleavage in the north/south or east/west direction in order to resolve the holliday junction • Heterodimer of RuvAB will work to recognize the holliday junction which will be cleaved by RuvC• Responsible for branch migration of holliday junctions • RuvA recognizes and binds to the holliday junction • RuvB is an ATP-dependent helicase • RuvC cuts the holliday junction randdomly choosing oritention (choice determines if there is a crossover) ▪ Cut north/south --> crossover ▪ Cut east/west --> patch ◦ RecG facilitates branch migration but doesn't have exonuclease activity and won't be able to cleave and will need to rely on the presence of previosus single strand breaks in the DNA in order for it to do the actual cutting Recombination in Meiosis • Homologous chromosomes must pair and undergo recombination priorto 1st meiotic division ◦ Pairing and recombination ensure proper alignment and segregation to daughter cells ◦ Generate diversity (arrangements of genes different from either parent) in the final products of meiosis ◦ Initiating by creating double stranded breaks Other types of Recombination • Site specific recombination ◦ Specific short sequences in both donor and recipient and special enzymes can direct recombination between sequences with little or no homology • Transposition ◦ Requires the ends of the transposable element and special enzymes◦ Allows the transposable element to be inserted at many differentsites without requiring homology • Illegitimate recombination ◦ Very low frequency recombination between non-homologous sequences Repair of DSBs • Homologous recombination (HR) uses the information in an intact double stranded DNA molecule to repair the broken DNA molecule◦ HR can repair a DSB perfectly• In eukaryotes, only cells in S or G2 have two copies of each chromosome so in many cells HR can't be used to fix a DSB• Nonhomologous end joining (NHEJ) can repair DSBs without needed anintact copy of the damaged DNA molecule ◦ The most important pathway for DSB repair in nonproliferating cells of higher eukaryotes ◦ CAN"T repair a DSB perfectly DSB Repair by NHEJ• NHEJ joins linear, double stranded DNA molecules end to end • No significant homology between the two ends is required • Ends are trimmed before joining --> NHEJ repair of DSB won't perfectly restore the original sequence• Telomeres protect chromosome ends from NHEJ• Bacteria the have NHEJ spend a lot of time in stationary phase (M. tuberculosis)• Simultaneous DSBs can result in translocationsMitosis • Account for nearly all of the cell divisions in your body • Chromosomal DNA is faithfully replicated and identical copies of the genome are passed along to both daughter cells Meiosis • Chromosomal DNA is subdivided unequally between daughter cells with some cells inheriting maternal alleles (red) and others inheriting paternal alleles (blue) • Only found in cells that can produce gametes/sex (germ cells)Alleles of different genes are inherited independently • Non-homologous chromosomes are sorted independently at meiosis • Homologous chromosomes undergo genetic recombination (=crossing over) during meiosis producing recombinant chromosomes containing a micture of meternal and paternal alleles ◦ Two genes located at oppposite ends of the same chromosome will also show independent inheritance Genetic Screen - method for discovering new genes • First an organism was exposed to radiation or chemical mutagens --> procedure was called mutagenesis (this kind is a random process every chromosome ,every cell experiences its own unique set of mutations ◦ Each offspring will inherit different mutations and they will be heterozygous for those mutations• Second the scientist identifies offspring with abnormal