The endonuclease IV family of apurinic/apyrimidinic endonucleases

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    Mutation Research 705 (2010) 217227

    Contents lists available at ScienceDirect

    Mutation Research/Review

    journal homepage: www.e lse.eContents

    1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217

    2. Sources and biological consequences of AP sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218

    3. Repair of AP sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218

    4. E. coli endonuclease IV. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219

    5. S. cerevisiae Apn1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222

    5.1. The role of Apn1 in mitochondrial BER . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223

    6. S. pombe Apn1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223

    7. C. elegans APN-1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224

    8. Conclusions and future directions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224

    Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225

    References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225

    1. Introduction

    Genomic DNA is constantly damaged by a variety of endoge-nous and exogenous agents. Base damage is one of the mostcommon forms of DNA damage. Oxidation, alkylation anddeamination of bases can disrupt base pairing and promotemutagenesis by leading to misincorporation errors during DNAreplication. It is therefore critical for cells to recognize andfaithfully repair these lesions to avoid cancer and premature aging.All kingdoms of life possess apurinic/apyrimidinic (AP) endonu-cleases, which participate in processing base lesions at various

    steps. AP endonucleases are named for their ability to cleave APsites in DNA, which are formed when DNA glycosylases cleavedamaged bases at the N-glycosidic bond. Some AP endonucleasesare also endowed with the ability to directly incise the DNA strandupstream of the primary lesion in a process termed nucleotideincision repair (NIR), leaving the damaged base on a 50 ap [1].Finally, AP endonucleases have 30 phosphodiesterase activity,which can process damaged 30 ends on single-strand breaks, and3050 exonuclease activity which can remove nucleotides from theend of a nick [2,3].

    The two families of AP endonucleases are named for theirarchetypal Escherichia colimembers, endonuclease IV (EndoIV) andexonuclease III (ExoIII). The two families are structurally unrelated,indicating that they evolved independently and not from acommon ancestor. Table 1 summarizes the key model organisms

    A R T I C L E I N F O

    Article history:

    Received 27 April 2010

    Received in revised form 3 July 2010

    Accepted 14 July 2010

    Available online 3 August 2010

    Keywords:

    Endonucleases

    Base excision repair

    Nucleotide incision repair

    Genomic stability

    Yeast

    C. elegans

    A B S T R A C T

    Apurinic/apyrimidinic (AP) endonucleases are versatile DNA repair enzymes that possess a variety of

    nucleolytic activities, including endonuclease activity at AP sites, 30 phosphodiesterase activity that canremove a variety of ligation-blocking lesions from the 30 end of DNA, endonuclease activity on oxidativeDNA lesions, and 30 to 50 exonuclease activity. There are two families of AP endonucleases, named for thebacterial counterparts endonuclease IV (EndoIV) and exonuclease III (ExoIII). While ExoIII family

    members are present in all kingdoms of life, EndoIV members exist in lower organisms but are curiously

    absent in plants, mammals and some other vertebrates. Here, we review recent research on these

    enzymes, focusing primarily on the EndoIV family. We address the role(s) of EndoIV members in DNA

    repair and discuss recent ndings from eachmodel organism in which the enzymes have been studied to

    date.

    2010 Elsevier B.V. All rights reserved.

    * Corresponding author. Tel.: +1 514 252 3400x4684; fax: +1 514 252 3430.

    E-mail address: dramotar.hmr@ssss.gouv.qc.ca (D. Ramotar).

    1383-5742/$ see front matter 2010 Elsevier B.V. All rights reserved.doi:10.1016/j.mrrev.2010.07.003Mini-review

    The endonuclease IV family of apurinic

    James M. Daley, Chadi Zakaria, Dindial Ramotar *

    Centre de Recherche, Hopital Maisonneuve-Rosemont, Universite de Montreal, 5415 de

    Communi ty address : wwwpyrimidinic endonucleases

    ssomption, Montreal, QC H1T 2M4, Canada

    s in Mutation Research

    vier .com/ locate / rev iewsmrlsev ier .com/ locate /mutres

  • J.M. Daley et al. /Mutation Research 705 (2010) 217227218in which EndoIV homologs have been identied and studied. InSaccharomyces cerevisiae, the EndoIV homolog Apn1 is responsiblefor the vastmajority of AP endonuclease activity in the cell [2,4]. AnEndoIV homolog (APN-1) also signicantly contributes to DNArepair in Caenorhabditis elegans [5]. In Schizosaccharomyces pombe,an EndoIV homolog exists but seems to play only a backup role inDNA repair [6]. EndoIV family members are also present in somesh (Danio rerio) and frogs (Xenopus tropicalis) but have not yetbeen studied in these organisms. EndoIV homologs are curiouslyabsent from higher organisms including insects and mammals.These organisms instead depend solely on ExoIII family members,such as human APE1, for AP endonuclease activity. Unlike EndoIV,ExoIII family members exist in all kingdoms of life. Like humancells, E. coli utilizes ExoIII as its major AP endonuclease, but alsoinduces EndoIV under stress conditions [79]. In contrast, the yeastExoIII homolog, Apn2, constitutes only aminor portion (
  • [(Fig._1)TD$FIG]

    lated

    ed b

    J.M. Daley et al. /Mutation Research 705 (2010) 217227 219endonuclease 1 (FEN-1) and, as in SP-BER, a DNA ligase seals thenick.

    AP sites can also be cleaved by AP lyases [48]. E. coliendonuclease III (EndoIII) is the prototypical AP lyase, and manyDNA glycosylases in higher organisms have additional AP lyaseactivity [4951]. Unlike AP endonucleases, AP lyases cleave on the30 side of the AP site through a b-elimination reaction to producean a,b-unsaturated aldehyde [5254]. This product blocks DNArepair synthesis and must therefore be removed by a 30

    phosphodiesterase before repair can proceed [11]. Most APendonucleases possess this activity and are capable of removinga variety of 30 blocking groups in addition to a,b-unsaturated

    Fig. 1.General model for AP endonuclease involvement in base excision repair and reor removed by glycosylases to form AP sites (right). The damaged base is resynthesiz

    are also repaired by the AP endonucleases.aldehydes, including 30 phosphates and 30 phosphoglycolates,which are present at single-strand breaks caused by oxidizingagents (Fig. 2B) [3,15,55]. Genetic data in E. coli and S. cerevisiaesuggests that AP sites are mainly processed by the AP endonu-cleases, with the AP lyases serving as a backupmechanism [50,5658]. The situation appears to be reversed in other organisms suchas S. pombe, however, where the lyase pathway predominates[59,60].

    4. E. coli endonuclease IV

    The rst indication regarding the presence of a specic class ofenzymes capable of processing AP sites came from studies in E. coli.ExoIII was the rst AP endonuclease discovered and accounts for90% of the total AP endonuclease activity in the cell [61]. ExoIIIwas rst described as a 30 to 50 exonuclease, and it was originallythought that the major cellular AP endonuclease activity was dueto a separate enzyme termed endonuclease II [62,63]. Later, it wasfound that the same mutation disrupts both exonuclease andendonuclease activities, and in 1976 the two activities wereattributed to the same enzyme, ExoIII [8,64]. ExoIII requires Mg2+

    and is therefore EDTA-sensitive [65]. ExoIII is also heat sensitive, asit is inactivated rapidly upon incubation of the puried protein atmoderately high temperatures [66,67]. ExoIII possesses three othercatalytic activities in addition to its AP endonuclease activity. It is a30 to 50 exonuclease specic for dsDNA, allowing it to degrade bluntends, 50 overhangs or nicks to produce segments of ssDNA [68].This activity is not observed on dsDNA with 30 overhangs that areover four bases long or on ssDNA [68]. ExoIII can also remove 30

    phosphates to generate 30 OH termini, and has RNase H activitythat degrades RNA in DNA-RNA hybrids [68].

    The hunt for a second AP endonuclease was initiated by thending that xth mutants lacking ExoIII still possess 10% of thecells total AP endonuclease activity [66,67]. EndoIV was isolatedfrom crude extracts derived from these mutants [66,67]. Thisenzyme can incise the phosphodiester bond 50 of AP sites andcertain oxidized nucleotides (NIR described below), but not othertypes of damage (e.g. bulky lesions), to create single-strand breakswith 30 OH termini [66]. Like ExoIII, EndoIV possesses 30

    pathways. Damaged bases can be incised to initiate nucleotide incision repair (left)

    y either short- or long-patch repair. Strand breaks with damaged termini (far right)phosphodiesterase activity allowing it to remove 30 blockinggroups at single-strand breaks such as those resulting from theactivity of AP lyases [69]. Puried EndoIV also possess 30 to 50

    exonuclease activity with a preference for 30 recessed ends ofdsDNA [70]. In contrast to the AP endonuclease and 30 phosphodi-esterase activities, this exonuclease activity is inhibited in thepresence of the metal chelator EDTA and reducing conditions [70].So far, a biological role for the exonuclease activity of EndoIV hasnot yet been dened. It is noteworthy that EndoIV expression isinduced asmuch as 20-fold by superoxide anion generators such asparaquat [9]. As such, the level of EndoIV becomes comparable tothat of ExoIII, but its 30 diesterase activity always remains lowerthan that of ExoIII [9,71]. Similarly, expression of the human ExoIIIhomolog APE1 is increased by oxidative stress [72].

    To date, E. coli EndoIV is the only member of the EndoIV familythat has been crystallized (Fig. 4A) [14,73]. The crystal structureshows that two of the Zn2+ ions coordinate a water molecule,which acts as the nucleophile to hydrolyze the DNA strand. Thethird Zn2+ ion stabilizes the resulting 30 OH. Mutation of themetal-coordinating residues impairs catalytic activity [73]. Otherunrelated nucleases utilize a similar mechanism to cleave DNA,but EndoIV is the only known DNA repair protein to employ thissystem [74,75]. Like many other DNA repair proteins, EndoIVrecognizes its substrate by ipping the AP site and its complemen-tary nucleotide out of the double helix (Fig. 4B) [14]. Instead ofkinking the DNA as shown for glycosylases, EndoIV bends the DNAat a 908 angle [14,76]. The increased exibility of AP site-

  • [(Fig._2)TD$FIG] J.M. Daley et al. /Mutation Research 705 (2010) 217227220containing DNA allows specicity for the target and prevents theenzyme from cleaving undamaged DNA [13]. Upon base ipping,EndoIV undergoes a conformational change that allows the highlyconserved residues Tyr72, Leu73 and Arg37 to form base stackinginteractions on either side of the AP site that stabilize the ipped-out conformation (Fig. 3, red arrows and Fig. 4C).Mutation of Tyr72to Ala decreases catalytic activity, but surprisingly does notprevent AP site ipping [73]. The space vacated by the ipped-outlesion (normally lled by Tyr72) is instead occupied by four watermolecules in the Tyr72Ala variant [73]. This suggests that Tyr72 isimportant for excluding solvent from the active site of the enzyme.

    Fig. 2. Chemical structures of EndoIV substrates, including (A) an AP site cleaved by thphosphodiesterase activity, and (C) cleavage upstream of a damaged base (a-20-deoxyThe active site can accommodate any base-free nucleotide,explaining the diverse phosphodiesterase activity of the EndoIVfamily on a variety of substrates.

    In the ExoIII family, crystal structures have been obtained forboth E. coli ExoIII and human APE1 [68,77]. Comparison withEndoIV reveals that the two families utilize completely different,independently evolved structures to accomplish the same task.Whereas EndoIV uses a TIM (triosephosphate isomerase) barrelfold consisting of eight a-helices and eight parallel b-strands tointeract with DNA, ExoIII and APE1 use a 4-layered a,b sandwichfold [68]. Both enzymes rely on the increased exibility of the AP

    e AP endonuclease activity, (B) a 30 phosphate blocking lesion removed by the 30

    adenosine is shown as an example) by the NIR activity.

  • [(Fig._3)TD$FIG] J.M. Daley et al. /Mutation Research 705 (2010) 217227 221site for recognition, however, and both probe the genome for APsites by inserting residues into the minor groove [13]. WhileEndoIV undergoes a striking conformational change upon AP sitebinding to accommodate the ipped-out nucleotide, APE1 doesnot; it is instead preformed to accept the AP site [77]. Bothenzymes cleave the DNA backbone by attacking the phosphodie-ster bond with a hydroxyl nucleophile, but different metals (Zn2+

    for EndoIV and Mg2+ for ExoIII) are used for charge neutralization.Thus, the two families of enzymes represent a striking example ofconvergent evolution, in which the two enzymes evolved to share

    Fig. 3. Alignment of EndoIV family members across multiple species. Red arrows indicateAP site. Blue arrows indicate conserved amino acids that coordinate the Zn2+ ion.many of the key aspects of the reaction despite starting from twocompletely different structures.

    The relative contribution of ExoIII and EndoIV to repair in vivowas investigated using mutants defective in either or both of thetwo enzymes. These studies revealed extensive overlap betweenthe two families [58]. Consistent with their respective activitiesin extracts, xthmutants lacking ExoIII are very sensitive to MMSwhereas nfomutants devoid of Endo...

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