Harpers

338 / CHAPTER 363′5′3′5′3′5′3′5′5′3′RECOGNITION AND UNWINDING5′3′OLIGONUCLEOTIDE EXCISIONBY CUTTING AT TWO SITES5′3′DEGRADATION OF MUTATED DNARESYNTHESIS AND RELIGATIONFigure 36–24. Nucleotide excision-repair. Thismechanism is employed to correct larger defects inDNA and generally involves more proteins than eithermismatch or base excision-repair. After defect recognition(indicated by XXXX) and unwinding of the DNA encompassingthe defect, an excision nuclease (exinuclease)cuts the DNA upstream and downstream of thedefective region. This gap is then filled in by a polymerase(δ/ε in humans) and religated.5′3′is also an important mechanism for repairing damagedDNA, such as occurs as a result of ionizing radiation oroxidative free radical generation. Some chemotherapeuticagents destroy cells by causing ds breaks or preventingtheir repair.Two proteins are initially involved in the nonhomologousrejoining of a ds break. Ku, a heterodimer of70 kDa and 86 kDa subunits, binds to free DNA endsand has latent ATP-dependent helicase activity. TheDNA-bound Ku heterodimer recruits a unique proteinkinase, DNA-dependent protein kinase (DNA-PK).DNA-PK has a binding site for DNA free ends and anotherfor dsDNA just inside these ends. It therefore allowsfor the approximation of the two separated ends.The free end DNA-Ku-DNA-PK complex activates thekinase activity in the latter. DNA-PK reciprocally phosphorylatesKu and the other DNA-PK molecule, on theopposing strand, in trans. DNA-PK then dissociatesfrom the DNA and Ku, resulting in activation of theKu helicase. This results in unwinding of the two ends.The unwound, approximated DNA forms base pairs;the extra nucleotide tails are removed by an exonuclease;and the gaps are filled and closed by DNA ligase.This repair mechanism is illustrated in Figure 36–25.Some Repair Enzymes Are MultifunctionalSomewhat surprising is the recent observation thatDNA repair proteins can serve other purposes. For example,some repair enzymes are also found as componentsof the large TFIIH complex that plays a centralrole in gene transcription (Chapter 37). Another componentof TFIIH is involved in cell cycle regulation.Thus, three critical cellular processes may be linkedthrough use of common proteins. There is also goodevidence that some repair enzymes are involved in generearrangements that occur normally.In patients with ataxia-telangiectasia, an autosomalrecessive disease in humans resulting in the developmentof cerebellar ataxia and lymphoreticular neoplasms,there appears to exist an increased sensitivity to damageby x-ray. Patients with Fanconi’s anemia, an autosomalrecessive anemia characterized also by an increased frequencyof cancer and by chromosomal instability, probablyhave defective repair of cross-linking damage.Ku and DNA-PK bindApproximationUnwindingAlignment and base pairingLigationPPFigure 36–25. Double-strand break repair of DNA.The proteins Ku and DNA-dependent protein kinasecombine to approximate the two strands and unwindthem. The aligned fragments form base pairs; the extraends are removed, probably by a DNA-PK-associatedendo- or exonuclease, and the gaps are filled in; andcontinuity is restored by ligation.