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Please give me your feedback with ICES evaluationNobel Prize in Chemistry-2015BER MR NERMCB 502A-2015. Lecture #14. Chromosomal damage and its Recombinational Repair— Classification of chromosomal lesions: types of "failure-by-design"— Chromosomal lesions are repaired by Rec-dependent processes— Homologous recombination: exchange by resolution of Holliday junctions— Genetics of Homologous Recombination— The principles of repair of chromosomal lesions— Biochemistry of recombinational repair— Major types of chromosomal lesions and corresponding recombinational repair pathways— Daughter-strand gap repair— When recombinational repair of daughter-strand gaps is impossible— Double-strand end repair— Replication restart— Chromosomal dimer resolution— When recombinational repair of double-strand breaks is unavailableClassification of chromosomal lesionsClassifica(on+of+chromosomal+lesions11+— … replication-dependent chromosomal lesions … … direct chromosomal lesions … — … group… into two major classes. — … tries to repair a crosslink… ("unhooking reaction"), … second strand is also damaged … — … blocked (unfillable) single-strand gap … DSBISCunhookingblocked ss-gapClassifica(on+of+chromosomal+lesions12+— … direct double-strand break… RFC… double-strand end. — … (RFC) … (can) generate two double-strand ends. — … nick in the terminus… (RFs) converging … one intact chromosome … double-strand break … — … blocked single-strand gaps (one strand interrupted)… double-strand ends (two strands interrupted). DSBClassifica(on+of+chromosomal+lesions13+— …. replication-independent … without strand interruptions are a chromosomal dimer and a DNA knot, … replication-dependent… locked replication fork and catenated sister chromosomes. — … classifying chromosomal lesions … number of DNA strands interrupted… direct or replication-dependent.catenanesA locked replication forkDNA knotNonfunctional DNA/chromosome structuresA classification of chromosomal lesions!# of strands Repair Interrupted Direct Replication-dependent mechanism 2 1 0 DSB RFD ISCL DSG dimer catenaneClassifica(on+of+chromosomal+lesions14+— … besides … interferance with the chromosome cycle… cannot be repaired by excision… — … depends on the intactness of the second DNA strand… either affect both DNA strands opposite each other … have no damaged DNA… — … look like natural design vulnerabilities (NDV), … … killing cells … via irreversible inactivation … . — … similar to zero-day vulnerabilities in programming.Zero-day vulnerability in computer software: … a disaster in waitingChromosomal lesions are repaired by Rec-dependent processes-1— … repairable at all? It looks like they are…:— … the mechanisms …? — … clueless … — … reach for Genetics … — In this case we could: 1) isolate … DNA-damage-sensitive mutants;2) … physical read-outs —> … unable to repair chromosomal lesions. — For example… sensitive to UV-light. UntreatedX-irradiated, 0'X-irradiated, 120'Chromosome separated in PFGEReturn to normalcy = repairChromosomal lesions are repaired by Rec-dependent processes-2— … epistatic analysis, … double mutants … single mutants… three major categories: 1) mutants deficient in photoreactivation (phr); 2) mutants deficient in the nucleotide excision repair (uvr); 3) rec mutants, … deficient in … homologous recombination:Group Primary Defect Removal of PD DSB / DSG repairphr photoreactivation +, by NER normaluvr nucleotide excision repair ±, by PhL and light normalrec homologous recombination ++, by NER and PhL defective— … phr and uvr mutants repair DSBs and DSGs no problem, but some rec mutants were completely deficient in chromosomal repair.Chromosomal lesions are repaired by Rec-dependent processes-3— For example,… lower molecular weight … in uvr mutants after UV-irradiation… —> daughter-strand gaps. — … further incubation … gradually becoming HMW… —> repair of daughter-strand gaps… — … stays LMW in the recA mutants, … Bottom (HMW)Bottom (HMW)Gradients are alkaline sucrose (ssDNA)Top (LMW)Top (LMW)No UV10' after UV120' after UVNo UV10' after UV120' after UVRec+ cells ∆recA cellsChromosomal lesions are repaired by Rec-dependent processes-4Recombination mutantsDefects in homologous recombinationDefects in repair of chromosomal lesionsChromosomal lesions are repaired by homologous recombinationHomologous recombination: exchange by resolution of Holliday junctions-1— What is homologous recombination? — … mostly identical in their sequences, … "homologous" … — … long chromosomes, … sisters, … (no) 100% identity, … biologically-relevant term. — HR is exchange between two chromosomes … Although originally "homology" was a qualitative term used by evolutionists to describe a common origin (like for these mammalian limbs), in molecular evolution, "homology" is used interchangeably with "sequence identity".Homologous recombination: exchange by resolution of Holliday junctions-2— A generic mechanism … separation and exchange … — … strands, when aligned reciprocally, … hybrid duplexes … "recombination intermediate”:5' 3'3' 5'5' 3'3' 5'5' 3'3' 5'5' 3'3' 5'5' 3'3' 5'5' 3'5'Recombination intermediateHybrid duplexesHolliday junctions3'(with the help of a topoisomerase)— … Holliday junctions, after Robin Holliday … significance. Robin HollidayHomologous recombination: exchange by resolution of Holliday junctions-3— … seen in EM …— … how to proceed … ? — … Holliday … "resolved" … pair of opposite strands. — … alternative ways … we isomerize .. by 180°. — … junction four-fold symmetrical, … . 5'3'5'3'5'3'5'3'5'3'5'3'5'3'5'3'Four-fold symmetryhttp://www.sciencedirect.com/science/article/pii/S0092867406014668Homologous recombination: exchange by resolution of Holliday junctions-4— … cleaved in either one … — … affects two out of four … — Both ways resolve … — The resulting nicks … sealed by DNA ligase. 5'3'5'3'5'3'5'3'ligaseligase5' 3'5'3'But, wait a minute, Robin….— … two such junctions … a single junction… 5' 3'3' 5'5' 3'5'Recombination intermediate3'— No worry, Holliday has thought of everything!3'3'Homologous recombination: exchange by resolution of Holliday
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