dna repair

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

DNA Repair


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 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


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


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



• Transcription-coupled pathway

– lesion detected by stalled RNA polymerase

– transcribed genes are highest priority

• Global pathway - slower, less efficient



• 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


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 & sealed by DNA ligase



• 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



• 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


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


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



• 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



• Another DSB repair pathway

– includes genetic recombination

– considerably more complex


DNA Replication and Repair


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


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


Unrepaired lesions block replication

• Polymerase stalls

• recruit specialized polymerase that is able to bypass the lesion

– thymidine dimer as example

– replicative polymerase (pol or ) replaced pol

– This enzyme inserts 2 A residues across from dimer

– XP-V mutation alters pol

– Cannot replicate past thymidine dimers


Unrepaired lesions block replication

• Polymerase 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)

dna repair

Documents

repair defectsner

dna ligaseber

dna backbonechemicals

dna repairstable

dna polymerase b

helicasesdamaged strand

uracil damage

new strand uncleardoes