Karp/CELL & MOLECULAR BIOLOGY 3E DNA Repair Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E

Karp/CELL & MOLECULAR BIOLOGY 3E Stable, but fragile Types of damage experience by DNA Ionizing radiation can break DNA backbone chemicals, some made by cell metabolism ultraviolet radiation: pyrimidine dimers thermal energy can depurinate adenine & guanine warm-blooded mammals lose ~10,000 bases/day Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E

Karp/CELL & MOLECULAR BIOLOGY 3E Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Figure 13.26

Karp/CELL & MOLECULAR BIOLOGY 3E Stable, but fragile Failure to repair causes mutations Can interfere with transcription and replication Can lead to malignant transformation Can speed aging It is essential that cells possess mechanisms for repairing this damage Repair mechanisms are extensive and efficient <1 base change per thousand escapes repair Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E

Karp/CELL & MOLECULAR BIOLOGY 3E Stable, but fragile Many repair proteins Repair is sometimes direct; but usually excised & replaced One enzyme uses sunlight energy to fix pyrimidine dimers Excision repair uses info in undamaged complementary strand DNA replication & repair share many parts & services Adverse effects seen in humans with repair defects Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E

NER = Nucleotide Excision Repair Works on bulky lesions like pyrimidine dimers & adducts Uses "cut-and-patch" mechanism 2 distinct NER pathways distinguished transcription coupled pathway slower global pathway Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E

NER = Nucleotide Excision Repair Transcription-coupled pathway lesion detected by stalled RNA polymerase transcribed genes are highest priority Global pathway - slower, less efficient Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E

NER = Nucleotide Excision Repair Damage recognition 2 NER pathways differ in lesion recognition subsequent repair steps are thought to be very similar TFIIH (participates in transcription initiation, too) A key component of repair machinery link between transcription & DNA repair two TFIIH subunits (XPB & XPD) are helicases damaged strand released by endonuclease cleavage (about 30 bases) gap filled by DNA polymerase, then ligase Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E

Karp/CELL & MOLECULAR BIOLOGY 3E Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Figure 13.27

BER = Base Excision Repair Base excision repair (BER) remove damaged bases alterations more subtle, distort the helix less Steps of BER DNA glycosylase removes base cleaves glycosidic bond holding the base to sugar "debased" deoxyribose phosphate removed combined action of an endonuclease & a phosphodiesterase Gap is then filled by DNA polymerase b & sealed by DNA ligase Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E

BER = Base Excision Repair Multiple DNA glycosylases each is more-or-less specific for a type of altered base Uracil - forms by hydrolytic removal of cytosine's amino group 8-hydroxyguanine - results from damage by oxygen free radicals 3-methyladenine - caused by alkylating agents Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E

BER = Base Excision Repair Uracil formation from cytosine explains why thymine used instead of uracil damage to cytosine = “normal” uracil uracil-DNA glycosylase is highly conserved protein E. coli & humans: 56% identity in amino acid sequence Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E

Karp/CELL & MOLECULAR BIOLOGY 3E Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Figure 13.28

Karp/CELL & MOLECULAR BIOLOGY 3E MMR = Mismatch Repair enzyme removes mismatched nucleotide in bacteria Parental strand has methyl-adenosine residues Provide signal for polarized repair removes & replaces from nonmethylated strand Returns correct base pair in eukaryotes the mechanism of identification of new strand unclear does not appear to use methylation signal Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E

Double-strand breakage repair Caused by ionizing radiation (X-rays, gamma rays) Also caused by chemicals (bleomycin, free radicals) Ultimately may prove lethal DSBs can be repaired by several alternate pathways Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E

Double-strand breakage repair NHEJ in mammalian cells non-homologous end joining the simplest & most commonly used complex of proteins binds to broken ends catalyzes a series of reactions that rejoin the broken strands mutants for NHEJ are very sensitive to ionizing radiation Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E

Karp/CELL & MOLECULAR BIOLOGY 3E Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Figure 13.29

Double-strand breakage repair Another DSB repair pathway includes genetic recombination considerably more complex Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E

DNA Replication and Repair Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E

Xeroderma pigmentosum (XP) inherited disease patients unable to repair damage from exposure to u.v. defect in 1 of 7 different genes nucleotide excision repair (NER) genes XPA, XPB, XPC, XPD, XPE, XPF & XPG Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E

Xeroderma pigmentosum (XP) patients susceptible to skin cancer via sun exposure capable of nucleotide excision repair only slightly more sensitive to UV light but, produced fragmented daughter strands after UV irradiation a variant form of XP, designated XP-V Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E

Unrepaired lesions block replication Polymerase stalls recruit specialized polymerase that is able to bypass the lesion thymidine dimer as example replicative polymerase (pol d or e) replaced pol h This enzyme inserts 2 A residues across from dimer XP-V mutation alters pol h Cannot replicate past thymidine dimers Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E

Unrepaired lesions block replication Polymerase h is member of a superfamily bypass polymerases are “error prone” trans-lesion synthesis (TLS) different basic structure from classic DNA polymerases they lack processivity: one or a few bases no proofreading capability humans have at least 30 TLS polymerases (genome project) Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E