DNA Repair

1. DNA Repair Karp/CELL & MOLECULAR BIOLOGY 3E

2. 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 Karp/CELL & MOLECULAR BIOLOGY 3E

3. Figure 13.26 Karp/CELL & MOLECULAR BIOLOGY 3E

4. 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 Karp/CELL & MOLECULAR BIOLOGY 3E

5. 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 Karp/CELL & MOLECULAR BIOLOGY 3E

6. 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 Karp/CELL & MOLECULAR BIOLOGY 3E

7. NER = Nucleotide Excision Repair • Transcription-coupled pathway • lesion detected by stalled RNA polymerase • transcribed genes are highest priority • Global pathway - slower, less efficient Karp/CELL & MOLECULAR BIOLOGY 3E

8. 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 Karp/CELL & MOLECULAR BIOLOGY 3E

9. Figure 13.27 Karp/CELL & MOLECULAR BIOLOGY 3E

10. 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 Karp/CELL & MOLECULAR BIOLOGY 3E

11. 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 Karp/CELL & MOLECULAR BIOLOGY 3E

12. 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 Karp/CELL & MOLECULAR BIOLOGY 3E

13. Figure 13.28 Karp/CELL & MOLECULAR BIOLOGY 3E

14. 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 Karp/CELL & MOLECULAR BIOLOGY 3E

15. 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 Karp/CELL & MOLECULAR BIOLOGY 3E

16. 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 Karp/CELL & MOLECULAR BIOLOGY 3E

17. Figure 13.29 Karp/CELL & MOLECULAR BIOLOGY 3E

18. Double-strand breakage repair • Another DSB repair pathway • includes genetic recombination • considerably more complex Karp/CELL & MOLECULAR BIOLOGY 3E

19. DNA Replication and Repair Karp/CELL & MOLECULAR BIOLOGY 3E

20. 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 Karp/CELL & MOLECULAR BIOLOGY 3E

21. 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 Karp/CELL & MOLECULAR BIOLOGY 3E

22. 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 Karp/CELL & MOLECULAR BIOLOGY 3E

23. 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) Karp/CELL & MOLECULAR BIOLOGY 3E