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Note for: Reverse genetic studies of the DNA damage response in the chicken B lymphocyte line DT40 (doi: 10.1016/j.dnarep.2004.03.039) Post-genomic era --> 1. reverse genetic tool is used to study the protein function 2. ppl using DT40 bc of many advantages; - efficient gene targeting - stable phenotype - lack p53 allow us to study the DNA damage due to the cells still can go through the interphase checkpoint even they have the error --  this is the reason why it is a good model to study the DNA damage and repair by observing on the chromosome. In higher eukaryote; 1. human and mouse, B-lymphocyte develop in bone marrow Functional IgV segments generate through site-specific process called V(D)J recombination 2. rabbit and chicken, B-lymphocyte develop in specific tissue; R-appendix of intestinal tract/C-bursa of Fabricius Functional IgV generate through intragenic homologous DNA recombination, called Ig gene conversion. Mature B-lymphocyte; human

Note for: Disruption of the BLM gene in ATM-null DT40 cells does not exacerbate either phenotype (doi: 10.1038/sj.onc.1207276 ) BLM and ATM; 1. autosomal recessive human disorder 2. immunodefficiency 3. genome instability 4. predisposition to develop cancer -- - BLM interact with ATM and recognize the abnormal DNA structures from the literature - in DT40; BLM+ATM-KO viable - no exacerbation of this KO is observed - in the end, conclude that each protein has "largely" distinct roles in recognizing the DNA lesion-but there are some parts overlap. ATM phosphorylates Nbs1 at Ser343-->stop Nbs to play role as lesion detection but change the function to be the adaptor and facilitating ATM-dependent phosphorylation of structural maintenance of structural maintenance of chromosome protein (SMC1)-->S-phase checkpoint ATM-->phosphorylate FANCD2 on Ser222--> it is suggested the role in ATM-FA damage response. Characteristic of BS-autosomal re

Note for: RAD18 and RAD54 cooperatively contribute to maintenance of genomic stability in vertebrate cells (doi: 10.1093/emboj/cdf534) Major pathway for post-replcational DNA damage; 1. TLS 2. HR Rad18- conserved from lower eukaryotes to vertebrates. double KO, synthetic lethal --> ach has nearly normal kinetic (in term of proliferation). Gap in this study; no one study the role of TLS in maintenance of chromosomal DNA. In yeast, S. cerevisiae; RAD6/RAD18 epistasis group (all in; 1. RAD6(UBC2) 2. RAD18 3. REV1 4. REV3 5. REV7 Rad6 -- E2 conjugating enzyme Rad18 -- has the DNA binding domain it is proposed that Rad18 recruited Rad6 and transferring Ub to the targeted proteins. Another gap --> so far, the targeted for Rad18+Rad6 is still unk, but it has been proposed that it could bind to DNA polymerase that play activity in TLS. Mammals, 2 homologs of RAD6 --> HR6A and HR6B 1 homolog RAD18 --> RAD18 AA comparison;

Note for: Effects of double-strand break repair proteins on vertebrate telomere structure (Nucleic Acids Res. 2002 Jul 1; 30(13): 2862–2870) Phenotype between mouse and DT40 on the telomere --> Ku(-/-) different At the very end; the authors conclude that "significant species-specific differences" in telomeric fn of DSB repair proteins. Telomeres are stable structure and invisible to DNA repair machinery, or else there will be the fusion of chromosome. Telomeres --> appeared as DNA-protein cpx. --> selective for telomerase to access but not end-DNA processing enzymes. There is the report showing that if there is no telomerase, telomere is maintained by recombination-based pw. Evidents showed that when KO genes in DNA repair group --> there is the defects in telomere structure; 1. telomere shortening 2. alterations to terminal DNA structure 3. end-to-end fusions Gap; They don't know how DNA repair protein involved with the

Note for: Nbs1 is essential for DNA repair by homologous recombination in higher vertebrate cells (doi: 10.1038/nature01125) It has been proposed that Rad50-Mre11-Nbs1 cpx possesses nuclease activity to create 3' single stranded tails. Mre11-Rad50 are very conserved among yeast and human, except Xrs2 (homolog to Nbs1,Nijmegen breakage syndrome,  in human also higher eukaryote). NBS1 -- part of the NRM cpx and responsible for ATP-driven DNA unwinding and nuclease activity. Mre1-null -- lethal in vertebrate cells, not yeast Rad50 null -- lethal in vertebrate cells, not yeast Nbs1 null is lethal in embryonic mice but not in DT40, therefore, we can study. Mitomycin C relies on HR-mediated post-replicational repair. NHEJ-end joining assay- 1. DT40 results in  29% microhomology 2. Ku70(-/-) - used 100% microhomology (lack of end-joining function) SCneo-reporter construct; 1. transiently transfected I-Sce I enzyme 2. DSB is introduced by I-Sce I

Note for: Werner and Bloom helicases are involved in DNA repair in a complementary fashion (doi:  https://doi.org/10.1038/sj.onc.1205143 ) Werner syndrome; 1. rare autosomal recessive disorder 2. premature senescence Bloom syndrome; 1. short stature 2. immunodeficiency 3. rare autosomal recessive common for these two; 1. chromosomal instability-->tumorigenesis WRN and BLM-->closely relate to RecQ helicase WRN and BLM - involve in DNA repair in a complementary fashion. Both BLM and WRN; 1. contain 7 signature motifs conserved in DNA/RNA helicases 2. BLM shows most similarity to RecQ subfamily of DNA helicases Recombinant WRN and BLM; 1. unwind duplex DNA in 3-5 direction with 3' overhang tail 2. both interact with RPA to facilitate the unwinding WRN has the exonuclease activity (N-terminal) which functions as proofreading during the DNA synthesis. BLM and WRN show very different profiles of sensitivity toward DNA damagin

Note for: Homologous DNA recombination in vertebrate cells (doi: 10.1073/pnas.111006398) Rad52 =RAD50, RAD51, RAD52, RAD54, RDH54/TID1, RAD55, RAD57, RAD59, MRE11, and XRS2. Primary structure is this group of protein is very conserved from yeast to human. This paper mentioned that though the gene structure is very conserved across the kingdoms but the phenotype regarding what has been observed in yeast cannot be implied in vertebrate. This review paper wants to point out the difference. even though, the DT40 cell is transformed by leukosis their property on gene conversion still remains. Not only DT40, other avian leukosis virus cell line-RP9 and v-rel-transformed cell lines-->high targeted as well. molecular mechanism for high targeting efficiencies are not clear. Observation; Gene that relates to gene conversion in Ig gene conversion might also promote the gene targeting efficiencies since these two processes use HR. Advantage of DT40; 1. i