Base excision repair initiation revealed by crystal structures and

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  • The EMBO Journal Vol.17 No.17 pp.52145226, 1998

    Base excision repair initiation revealed by crystalstructures and binding kinetics of human uracil-DNAglycosylase with DNA

    Sudip S.Parikh, Clifford D.Mol,Geir Slupphaug1, Sangeeta Bharati1,Hans E.Krokan1 and John A.Tainer2

    The Skaggs Institute for Chemical Biology, Department of MolecularBiology, MB-4, The Scripps Research Institute, 10550 North TorreyPines Road, La Jolla, CA 92037-1027, USA and1UNIGEN Center forMolecular Biology, The Norwegian University of Science andTechnology, N-7005 Trondheim, Norway2Corresponding authore-mail: jat@scripps.edu

    Three high-resolution crystal structures of DNA com-plexes with wild-type and mutant human uracil-DNAglycosylase (UDG), coupled kinetic characterizationsand comparisons with the refined unbound UDG struc-ture help resolve fundamental issues in the initiationof DNA base excision repair (BER): damage detection,nucleotide flipping versus extrahelical nucleotidecapture, avoidance of apurinic/apyrimidinic (AP) sitetoxicity and coupling of damage-specific and damage-general BER steps. Structural and kinetic results sug-gest that UDG binds, kinks and compresses the DNAbackbone with a SerPro pinch and scans the minorgroove for damage. Concerted shifts in UDG simul-taneously form the catalytically competent active siteand induce further compression and kinking of thedouble-stranded DNA backbone only at uracil and APsites, where these nucleotides can flip at the phosphatesugar junction into a complementary specificity pocket.Unexpectedly, UDG binds to AP sites more tightly andmore rapidly than to uracil-containing DNA, and thusmay protect cells sterically from AP site toxicity.Furthermore, AP-endonuclease, which catalyzes thefirst damage-general step of BER, enhances UDGactivity, most likely by inducing UDG release via sharedminor groove contacts and flipped AP site binding.Thus, AP site binding may couple damage-specific anddamage-general steps of BER without requiring directproteinprotein interactions.Keywords: abasic sites/crystal structure/DNA repair/proteinDNA interactions

    Introduction

    Cells endure incessant DNA base damage which threatenstheir viability and genomic stability (Lindahl, 1993). DNAbase excision repair (BER) is a key pathway for restoringthe chemical integrity of DNA and, in humans, requiresthe action of a minimum of four enzymes: a damage-specific DNA glycosylase, followed by the BER generalapurinic/apyrimidinic (AP)-endonuclease, DNA poly-merase and DNA ligase (Kubotaet al., 1996; Nichollet al., 1997; Parikhet al., 1997). Detection and removal

    5214 Oxford University Press

    of base lesions by damage-specific DNA glycosylasesinitiates BER and creates AP sites in DNA. In humans,the principal AP-endonuclease (HAP1) cleaves the phos-phodiester bond 59 of AP sites regardless of the originalbase lesion, priming repair synthesis by DNA polymerase (Pol). Pol inserts the correct nucleotide and DNAligase restores the phosphodiester bond. Thus, BER occursin two stages: the initial, damage-specific stage carriedout by individual DNA glycosylases targeted to distinctbase lesions, and a damage-general stage that processesthe resulting central AP site intermediates.

    Approximately 10 000 AP sites per day are generatedin each human cell, both spontaneously and by DNAglycosylases (Lindahl, 1993). However, AP sites areunstable, autolytically degrading into abnormal DNAstrand breaks (Lindahl, 1990) and disrupting several cellu-lar processes. AP sites retard DNA polymerase, causebase misincorporation (Klinedinst and Drinkwater, 1992;Xiao and Samson, 1993) and are highly mutagenic at thelevel of transcription (Zhou and Doetsch, 1993). Moreover,AP sites engage in suicide reactions with topoisomeraseI, leading to permanent DNA damage and prematurecell death (Pourquieret al., 1997), and form covalentcomplexes with topoisomerase II that cause DNA double-strand breaks (Kingma and Osheroff, 1997), whichcan bind poly(ADP-ribose) polymerase and corruptcheckpoints between cell cycle arrest and apoptosis(Prasadet al., 1996). Thus, DNA glycosylases present abiological paradox: their product AP site is far morecytotoxic when left exposed within cells than the originalbase damage.

    Although the damage-general BER enzymes (HAP1,Pol and DNA ligase) interact directly with each other(Caldecott et al., 1995; Kubotaet al., 1996; Bennettet al., 1997), and possibly within a multi-enzyme complex(Prasadet al., 1996), there is no evidence for the directinteraction of any of these enzymes with the damage-specific DNA glycosylases. It therefore becomes importantto identify possible means of coupling the base damage-specific DNA glycosylases and the subsequent damage-general steps of BER.

    Uracil-DNA glycosylase (UDG) is particularly suitablefor this investigation in several respects. As the firstknown DNA glycosylase (Lindahl and Nyberg, 1974),UDG is biochemically (Varshney and van de Sande, 1991;Slupphauget al., 1995) and structurally (Molet al.,1995a,b; Savvaet al., 1995; Slupphauget al., 1996) wellcharacterized, and is a prototype for other DNA repairglycosylases. Furthermore, its fundamental biologicalimportance is evidenced by its conservation from bacteriato humans and even some viral pathogens (Molet al.,1998). The major biological function of UDG is to excisefrom DNA the uracil produced by cytosine deamination(Slupphauget al., 1995). Based on the 2.9 A resolution

  • DNA base excision repair initiation

    crystal structure of a catalytically impaired double mutantUDG bound to DNA, UDG is hypothesized to flip uracilnucleotides out of the DNA base stack using a pushpull mechanism in which a leucine side chain penetratesinto the DNA (push) and complementary interactions fromthe uracil recognition pocket facilitate final productivebinding (pull) (Slupphauget al., 1996). However, nostructure is available for leucine-containing wild-typeUDG bound to its biological substrate DNA. Furthermore,the potential mechanisms by which UDG initially findsuracil bases in DNA, acts in reducing AP site toxicityand is coordinated with subsequent BER steps remainmysterious.

    Thus, four key questions regarding DNA glycosylasestructure and function remain paramount for understandingBER. What governs initial DNA base lesion detection byglycosylases? Does enzyme binding facilitate nucleotideflipping or represent extrahelical base recognition? Afterglycosylic bond cleavage, how is the cell protected fromthe mutagenic and apoptotic effects of the AP site product?How might the damage-specific and damage-general stagesof BER be coordinated? To address these issues, wedetermined and analyzed high-resolution co-crystal struc-tures of the biologically relevant trapped product com-plexes of human wild-type UDG with UG- and UA-containing double-stranded (ds)DNA, as well as of acatalytically impaired mutant UDG bound to DNA con-taining a flipped-out AP site. These UDGDNA structuressuggest an efficient processive mechanism for uracildetection in DNA, support active nucleotide flipping,reveal the structural basis for preferential enzymaticexcision of uracil from UG mispairs rather than UA pairsand establish that UDG can flip AP sites out of DNA andbind them specifically. Measured DNA-binding kineticsand affinities of wild-type and mutant UDGs for biologic-ally relevant dsDNA targets support our new mechanismfor damage detection and demonstrate tight binding ofUDG to AP sites in DNA. We further show that UDGactivity is enhanced by the AP-endonuclease HAP1, sug-gesting indirect coupling of BER stages. Together, theseresults support a new, comprehensive model for BERinitiation that couples damage-specific DNA glycosylasesand subsequent damage-general stages to avoid the toxicityof AP sites in DNA. We propose that damage-specificDNA glycosylases, like UDG, bind or rebind product APsites until release is induced by shared DNA minorgroove and AP site interactions with the damage-generalAP-endonuclease, thus initiating the subsequent BERsteps.

    Results and discussion

    Leu272 and enzyme activityWild-type human UDG (wtUDG) excises uracil fromdsDNA with a marked preference for UG mispairs, andthe efficiency of this uracil excision is dependent onLeu272. Replacement of Leu272 with alanine (L272A)alters enzyme activity on both UG mispairs and UA basepairs (Figure 1). The effect of residue 272 on catalysis issurprising as it extends from a protruding loop.10 from the uracil recognition pocket such that a mutation atthis position would not interfere directly with uracilbinding (Mol et al., 1995; Slupphauget al., 1996).

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    Fig. 1. Uracil excision from UA base pairs and UG mispairs by wild-type and L272A UDGs. Enzymes were incubated with 19 bpoligonucleotides (59-TGAAATTGUTATCCGCTCA-39) containingeither a central UG mispair (dashed lines) or a UA base pair (solidlines) and assayed for uracil excision at the indicated time points forwtUDG (diamonds) or L272A (circles). Excision by L272A can bedetected at longer times or elevated substrate concentrations. Resultsare the average of three independent experiments with standarddeviations within 10%.

    However, the L272A mutant of human UDG is severelyimpaired, with uracil excision efficiencies,1% that ofwild-type for both substrates (Figure 1). These datatherefore argue directly against the recent hypothesis thaturacil binding dominates nucleotide flipping by all UDGs,which was proposed from studies of viral UDG (Panayotouet al., 1998). Excision data, however, do not distinguishwhether the reduction in activity for L272A is due todeficient DNA binding, uracil detection, nucleotide flip-ping or product release. Establishing these properties ofUDG is key to understanding BER initiation by damage-specific glycosylases.

    UDGDNA co-crystal structuresTo help establish the role of residue 272 and the propertiesof UDG acting in damage-speci

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