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

2. 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

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

4. 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

5. 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

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

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

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

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

14. 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

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

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

18. 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

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