MCB Exam 3Lecture 20—DNA RepairDNA Repair in Bacteria1. Mismatch Repair (MMR)—fixes mismatches resulting from replication errors2. Base Excision Repair (BER)—Udg (uracil DNA glycosylase) in bacteria, UNG (uracil N-glycosylase) in humans, removes U from DNA, removes modified basesfrom DNA3. Nucleotide Excision Repair (NER)—removes the wrong base along with adjacentnucleotides4. SOS-Translation DNA Synthesis—when all else fails, put in anything, alternative DNA polymerases, error prone replication 5. Recombinational Repair *Genetic crossing over 6. Non-homologous End Joining (NHEJ) (meiosis) & repair of DSBs*Meiosis- Cell division, occurs in preparation for sexual reproduction, the diploid genome ofsomatic cells is reduced to the haploid genome of gametes- Homologous Recombinationo When crossing over occurs between maternal and paternal chromosomes,the result is the formation of recombinant chromatids that contain a mix of maternal and paternal alleleso During prophase of Meiosis I a pair of homologous chromatids may crossover and undergo genetic recombinationo Adaptive feature that helps increase the genetic diversity of the speciesDouble-Stranded Breaks (DSBs)- HR is widely conserved mechanism for repairing DSBs in chromosomal DNA- Unusually high rate of crossovers resulting from DSB repair of mitotically growingcells with a damaging agent (bromodeoxyuridine)- Extremely dangerous, broken ends subject to nucleases, chromosomes fragments lost during cell division- Single-stranded break (nick) will become a DSB if DNA replication occurs before it is repairedHomologous Recombination- Recombination between DNA molecules whose base pair sequence is nearly identical- Critical mechanism for repair of mutations that involve double-stranded breaks (DSBs) - Found in all organisms, enables exchange of genes among closely related organisms- Other kinds of recombination:o Site-specific: depends on homology btn much shorter seuqnces in both donor and recipient and special enzymes that direct the recombination, immunoglobin gene arrangements, integration of lysogenic bacteriophages into genomso Transposition: means for transposons to be inserted into any DNAo Illegitimate: occurs at low frequency between non-homologous sequences, non-homologous end joiningRecombination- Homologous recombination begins when a double strand break (DSB) is introduced into one of a pair of aligned homologous chromosomes- Processing to generate gapped DNA with 3’ overhangs- Initiationo Highly conservedo Strand invasion of 3’ endo Requires DNA synthesis by DNA Pol I (5’3’)- Branch Migrationo Junction that slides along the chromosome after holliday junctions have formedo RuvAB (in E. coli)—responsible for branch migration of Holliday Junctions RuvA—tetramer recognizes and binds to the Holliday Junciton RuvB—ATP-dependent helicase that unwinds the DNA- Holliday Junctiono Physically join chromosomes, and will not allow them to separate during cell divisiono To allow for cell dibision, each Holliday Junction must be resolved by cleaving and religating two of the four DNA strandso Resolution By cleaving in either of two different orientations RuvC—randomly cleaves 2 of the DNA strands 2 Orientationsa. Splice/Crossover products reassortment of flanking genes, Holliday junction switches the two end of the orginial chromosome pairb. Patch or noncrossover products, no reassortment Both modes leave a patch of heteroduplex at the site of the junctionRepairing a Collapsed Replication Fork- Replication through a single-strand break (nick) will collapse the replication fork- HR can re-establish replication by a similar process to DSB repair- Two ends of the DSB will invade the intact chromosome and anneal with its two strands- Fork will be repaired following Holliday junction resolution and the replisome can renew itselfHomologous Recombination in E. coli- More than one pathway- Focus on primary HR pathways- Different proteins for recombination process:o RecA—required for all pathwayso RecBCD—important for DSB repairo RuvABCo Ss DNA binding protein, DNA Pol I, DNA ligase- RecAo Mutants in E. coli are incapable of HRo Widely conserved among all cells, eukaryotic homolog—Rad51o Initially binds to a ss and forms filament 5’3’, then searches for homology and initiates strand invasion and duplex formationo Requires at leas 50 bp of homologous sequence for successful strand invasion, but more efficient with longer regions of homologyo Polymerizes (forms filaments) on ssDNA **o Strand invasion is mediated by the RecA-coated 3’ end of the double-stranded break- Repair of DSBs mediated by RecBCD:o Generate a 3’ overhang for strand invasiono RecB—has a 3’5’ helicase and nuclease activityo RecD—has a 5’3’ helicase activityo Binds to the DSB, helicase unqinds the DNA, and the nuclease activity degrades both strands from the DSB, but the 3’ end is shortned more slowly- When the RecBCD encounters a Chi (X) site, it stops degrading the 3’ strand altogether and becomes more active degrading the strand with the 5’ end, this generates the 3’ overhang needed to initiate strand invasion and recombination- RecC—recognizes the Chi site- Chi (X) site—over represented in E. coli genome, crossover hotspot instigator- Too much repair? Inceased U incorporation leads to increased attempted repair, Ung and BER, death, and in the absence of recombination this ds break is lethal Repair of Double-Stranded Breaks- HR uses the info in an intact ds DNA molecule to repair the broken DNA molecule, HR can repair breaks perfectly with essentially no loss of sequence info- In eukaryotes, only cells in S or G2 phase have completed replication and have 2copies of each chromosome, prior to replication, HR cannot be used to fix a DSBNonhomologous End Joining (NHEJ)- Alternative method to repair DSCs without the need for an intact copy of the damaged DNA molecule- Cannot repair a DSB perfectly, loses sequence info- Most important pathway for DSB repair in the non-proliferating cells of higher eukaryotes- Joins linear dsDNA molecules end to end, no significant homology btn the two ends is required- Ku—recognizes the DSB- DNA-PKcs—protein kinase that recruits Artemis- Artemis—multi-functional nuclease that processes the ends- Ligase complexed to some other proteins joins the ends- Various DNA polys may be required to fill in gaps- Ends are trimmed