Transcript ch13c

DNA Repair
Copyright, ©, 2002, John Wiley & Sons, Inc.,
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
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Figure 13.26
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
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
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
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Figure 13.28
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
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