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DNA Repair 52 (2017) 1–11

Contents lists available at ScienceDirect

DNA Repair

j ourna l ho me pa ge: www.elsev ier .com/ locate /dnarepai r

role for the base excision repair enzyme NEIL3 ineplication-dependent repair of interstrand DNA cross-links derivedrom psoralen and abasic sites

hiyu Yanga, Maryam Imani Nejada, Jacqueline Gamboa Varelaa, Nathan E. Pricec,insheng Wangc, Kent S. Gatesa,b,∗

University of Missouri Department of Chemistry, 125 Chemistry Building Columbia, MO 65211, United StatesUniversity of Missouri Department of Biochemistry, 125 Chemistry Building Columbia, MO 65211, United StatesUniversity of California-Riverside, Department of Chemistry, 501 Big Springs Road Riverside, CA 92521-0403, United States

r t i c l e i n f o

rticle history:eceived 12 February 2017ccepted 13 February 2017vailable online 20 February 2017

eywords:NA cross-linkross-link repair

a b s t r a c t

Interstrand DNA–DNA cross-links are highly toxic lesions that are important in medicinal chemistry,toxicology, and endogenous biology. In current models of replication-dependent repair, stalling of areplication fork activates the Fanconi anemia pathway and cross-links are “unhooked” by the actionof structure-specific endonucleases such as XPF-ERCC1 that make incisions flanking the cross-link. Thisprocess generates a double-strand break, which must be subsequently repaired by homologous recombi-nation. Recent work provided evidence for a new, incision-independent unhooking mechanism involvingintrusion of a base excision repair (BER) enzyme, NEIL3, into the world of cross-link repair. The evidence

anconi anemiaomologous recombinationase excision repair

suggests that the glycosylase action of NEIL3 unhooks interstrand cross-links derived from an abasic siteor the psoralen derivative trioxsalen. If the incision-independent NEIL3 pathway is blocked, repair revertsto the incision-dependent route. In light of the new model invoking participation of NEIL3 in cross-link

PF-ERCC1EILbasic sitesoralen

repair, we consider the possibility that various BER glycosylases or other DNA-processing enzymes mightparticipate in the unhooking of chemically diverse interstrand DNA cross-links.

© 2017 Elsevier B.V. All rights reserved.

ontents

1. Introduction. DNA Interstrand cross-links in biology and medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22. Replication-dependent cross-link repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23. Base excision repair DNA glycosylases and their involvement in cross-link repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24. Evidence for a direct role for the catalytic action of a BER glycosylase in cross-link repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35. The glycosylase NEIL3 is involved in unhooking psoralen and Ap-derived interstrand cross-links . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46. Comparison of incision-dependent and incision-independent cross-link repair pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57. New possibilities suggested by the role of NEIL3 in cross-link repair: considering other enzyme activities that could catalyze replication-dependentunhooking of interstrand cross-links . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .68. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Abbreviations: BCNU, carmustine or 1,3-bis(2-chloroethyl)-1-nitrosourea; BER, basranslesion synthesis; HR, homologous recombination; NER, nucleotide excision repair; Fdndonuclease; Fapy, formamidopyrimidine; LC–MS/MS, liquid chromatography–tandem

∗ Corresponding author at: University of Missouri Department of Chemistry, 125 ChemE-mail address: gatesk@missouri.edu (K.S. Gates).

ttp://dx.doi.org/10.1016/j.dnarep.2017.02.011568-7864/© 2017 Elsevier B.V. All rights reserved.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

e excision repair; CMG helicase, Cdc45-MCM-GINS helicase; NEIL, Nei-like; TLS,T, 2′-deoxy-2′-fluoroarabinofuranosyl thymine; Ap, DNA abasic site; APE, apurinic

mass spectrometry.istry Building Columbia, MO 65211, United States.

2 Repair 52 (2017) 1–11

1m

sscnaDoftcgieintudtt

2

clcAGchooar[tCBc−ie[taiaietusdmct(bca

Fig. 1. Model for replication-dependent interstrand cross-link repair. (a) CMG heli-case collides with cross-link and dissociates. (b) Structure-specific endonucleasesunhook the cross-link. (c) Homologous recombination repairs double-strand break(top duplex). Possible nuclease trimming of the oligonucleotides flanking the adduct,

Z. Yang et al. / DNA

. Introduction. DNA Interstrand cross-links in biology andedicine

Reading and replicating the genetic information encoded in theequence of nucleobases in DNA requires cellular operations thateparate the two strands of the double helix [1–3]. Interstrandross-links introduced by bifunctional alkylating agents such asitrogen mustard anticancer drugs [4–7] prevent strand separationnd compromise critical cellular functions of DNA [8,9]. As a result,NA cross-links are highly toxic to cells [10,11]. A wide range ofrganisms, from bacteria to mammals, possess elaborate systemsor the repair of interstrand cross-links [12]. Over twenty pro-eins are required for cross-link repair in vertebrates [12–16]. Theonserved nature of such resource-intensive repair systems sug-ests that the formation of interstrand cross-links in cellular DNAs an inevitable fact of life. Endogenous processes or unavoidablexposure to environmental agents have the potential to generatenterstrand cross-links in cellular DNA [8,17–27], but the chemicalature of the selection pressures driving the evolution and reten-ion of cross-link repair systems across all walks of life remainsncertain. In medicine, the repair of interstrand cross-links helpsefine response and resistance to drugs such as cisplatin, carmus-ine (BCNU), and nitrogen mustards that are commonly used in thereatment of human cancers [28–33].

. Replication-dependent cross-link repair

When DNA replication is stalled by an interstrand cross-link,omplex repair processes are activated [34]. There are excel-ent comprehensive reviews of replication-dependent interstrandross-link repair [12–16] and we will summarize briefly here.dvancement of a replication fork is stalled when the Cdc45-MCM-INS (CMG) helicase complex [2,35,36] encounters an interstrandross-link. Due to the size of this complex, DNA polymerizationalts between −40 and −20 nucleotides (nt) from the 3′-sidef the cross-link on the leading strand (Fig. 1) [34,37]. Stallingf a replication fork activates the Fanconi anemia pathway andssociation of the FANCD2/FANCI heterodimer with the stalledeplication fork leads to ubiquitination of this protein complex34,38–45]. An emerging view holds that cross-link repair is ini-iated when two replication forks converge at the lesion [37]. TheMG helicases then dissociate in a process that involves BRCA1-ARD1 and ubiquitin signaling [46]. Dissociation of the helicaseomplex [2,35] enables DNA polymerization to progress to the1 position immediately preceding the cross-link on the lead-

ng strand (Fig. 1). The activated FANCD2/FANCI complex recruitsndonucleases that make incisions on either side of the cross-link38,41,45]. In this process, the SLX4 protein serves as a scaffoldhat coordinates the action of several structure-specific endonucle-ses including XPF-ERCC1, MUS81-EME1, and SLX1 [45,47,48]. Thenvolvement of XPF-ERCC1 has been clearly demonstrated [49,50]nd cells deficient in these proteins are acutely hypersensitive tonterstrand cross-linking agents [44,51–54]. The identity of otherndonucleases that make incisions during cross-link repair, andhe circumstances under which they become involved, remainsnder investigation [45,48,51,52,55–58]. Incisions by structure-pecific endonucleases “unhook” the cross-link and generate aouble strand break (Fig. 1). The unhooked cross-link remnantay be trimmed [34] to a smaller adduct (possibly by the exonu-

lease activity of SNM1A) [51,59] to facilitate lesion bypass byranslesion synthesis (TLS) polymerases such as Pol� (kappa), Pol�

eta), and Pol � (nu) [60–63]. Extension may then be carried outy a complex containing REV1 and Pol� (zeta) [34,64,65]. Indeed,ells deficient in REV1 and Pol� are hypersensitive to cross-linkinggents [64,66]. It remains uncertain whether REV1/Pol� cooperate

TLS, and extension past the adduct remnant (bottom duplex). (d) Cross-link remnantmay be removed by NER.

with other TLS polymerases as seen in the bypass of pyrimidinedimers [67]. Homologous recombination (HR) using the newly syn-thesized duplex on the leading strand as the homology repairpartner is required to repair the double-strand break [68–71].Finally, nucleotide excision repair (NER) may remove the unhookedcross-link remnant to regenerate a native DNA duplex [70]. Thereis much evidence supporting the general pathway shown in Fig. 1;however, it is worth noting that a distinct pathway involving repli-cation traverse of a psoralen-derived cross-link without repair hasbeen suggested [72,73].

3. Base excision repair DNA glycosylases and theirinvolvement in cross-link repair

The base excision repair (BER) pathway is critical for the removal

of damaged, misincorporated, and mispaired DNA bases [74–81].BER typically begins with DNA glycosylase enzymes that recognizea damaged or mispaired nucleobase (B* in Fig. 2A) and catalyzehydrolysis of the glycosidic bond holding the base to the deoxyri-

Z. Yang et al. / DNA Repair 52 (2017) 1–11 3

Fig. 2. Base excision repair enzymes remove damaged or mispaired nucleobases from DNA. Panel A: The mechanism of base removal by BER enzymes involves hydrolysis ofthe glycosidic bond. Shown here is a dissociative hydrolysis mechanism involving an oxocarbenium ion intermediate. Panel B: BER enzymes typically act on DNA substratesin which the damaged nucleobase is extruded from the double helix. The image shows the DNA substrate in a DNA-hOGG1 complex (protein structure omitted) in whicht u). Par substg

bos“–aeiDe5n

iiaHplpoctamptfgTbiBtrFi

he 8-oxo-G base is extruded from the duplex (image prepared from pdb entry 3ktesistant to hydrolysis by BER enzymes because the electron-withdrawing fluoro

lycosylase reaction.

ose backbone [82]. Various BER glycosylases remove a wide arrayf damaged and mispaired nucleobases [74–80]. Importantly, sub-trate recognition and catalysis by glycosylases typically involveflipping out” (extrusion of) the target base from the double helix

a process that is presumably precluded for bases involved inn interstrand cross-link (Fig. 2B) [81–83]. Monofunctional BERnzymes catalyze hydrolytic removal of a nucleobase leaving anntact abasic (Ap) site in the DNA (Fig. 2A), while bifunctionalNA glycosylases remove the base and also catalyze a subsequentlimination (lyase) reaction that generates a strand break with a′-phosphoryl end group and 3′-deoxyribose phosphate sugar rem-ant [74,76,78,79,82,84–86].

Over the years, there have been reports that BER proteins cannteract with cross-link repair pathways [87]. For example, theres evidence that murine 3-methyladenine glycosylase (Aag) plays

role in cellular resistance to psoralen-derived cross-links [88].owever, in vitro studies showed that human AAG did not bind orrocess a 21 base pair duplex containing a psoralen-derived cross-

ink, leading the authors to conclude that “Aag’s role in conferringrotection against psoralen-induced (cross-links) is either indirectr involves a DNA substrate that is an intermediate of the repair pro-ess” [88]. In a separate example, the glycosylase NEIL1 was showno accumulate at psoralen-derived interstrand cross-links in cellsnd inhibit repair of these lesions [89]. In vitro gel electrophoreticobility shift assays demonstrated that NEIL1 bound to a 21 base

air cross-linked duplex, with an affinity at least 4-fold greater thanhat for a native control duplex [89]. In another set of studies, it wasound that the deletion of the BER proteins Pol � and uracil DNAlycosylase (UNG) conferred resistance to cisplatin toxicity [90].his led to a proposal that UNGs remove uracil residues generatedy deamination of cytosine residues near cisplatin cross-links, thus

nhibiting subsequent repair [91]. In a final early example whereER may influence the repair of cross-links, Couvé et al. showed

hat NEIL1 can excise an unhooked, psoralen-derived cross-linkemnant from a three-stranded structure (e.g. bottom duplex inig. 3) [92,93]. This type of glycosylase activity would not providenitial unhooking of the cross-link, but could provide an alternative

nel C: Damaged bases attached to 2′-deoxy-2′-fluoroarabinofuranosyl residues areituent on the sugar destabilizes the oxocarbenium ion-like transition state of the

to NER for the final “clean-up” of the psoralen cross-link remnant(last step in Fig. 3).

4. Evidence for a direct role for the catalytic action of a BERglycosylase in cross-link repair

A recent study by Walter and coworkers provides evidence thatthe catalytic activity of a BER glycosylase enzyme can play a centralrole in the repair of interstrand DNA cross-links [94]. In this work,the direct catalytic action of the BER glycosylase NEIL3 was impli-cated in the repair of both psoralen-derived and abasic site-derivedinterstrand DNA cross-links in Xenopus egg extracts.

Psoralens are a class of plant-derived DNA cross-linking agents[95]. The planar furocoumarin system intercalates between thebase pairs of duplex DNA where 300–400 nm light can inducereactions between the pi-bonds of the natural product and the5,6-double bonds of thymine and cytosine residues to gen-erate both covalent monoadducts and interstrand cross-links(Fig. 4A) [96–99]. Interstrand cross-links arise preferentially at5′-TA sequences [100] via sequential photoinduced [2 + 2] cycload-ditions of thymine residues with the 4′,5′-double in the furan ringand the 3,4-double bond of the pyrone system [95,98] (Fig. 4B). DNAduplexes containing a psoralen cross-link display local unwind-ing at the cross-link site but, overall, retain a B-form structure[96–102]. However, the cross-link structure in duplex DNA maynot be directly relevant to the X-shaped and Y-shaped structuresgenerated at a stalled replication fork, which are the true substratesfor replication-coupled repair [48].

In the Xenopus egg extract system of Semlow et al., the repairof a psoralen cross-link was distinct from that previously observedfor a cisplatin interstrand cross-link [94]. Relatively small amountsof homologous recombination intermediates were seen and, whileFANCD2 was ubiquitinated, depletion of the FANCD2/FANCI com-

plex had a minimal effect on repair. This led the authors to considerthe possibility that the psoralen cross-link might be unhooked bythe action of a BER glycosylase. This process would generate anabasic site on one strand and a modified thymine residue on the

4 Z. Yang et al. / DNA Repa

Fig. 3. Model for replication-dependent, incision-independent repair of a psoralen-derived interstrand cross-link. (a) CMG helicase stalls at cross-link. (b) NEIL3unhooks the cross-link. (c) TLS and extension past the Ap site and psoralen adductremnant. (d) Repair of the Ap site by a pathway involving APE, pol �, and ligase III(d

oDttobm(lFoogt

tii

rated thymine rings in the psoralen cross-link resemble the known

upper duplex) and NER or BER to remove the psoralen cross-link remnant (loweruplex).

ther (Fig. 3). Several lines of evidence supported this supposition.epletion of the TLS polymerase REV1 caused stalling of replica-

ion at or near the −1 position on both strands. Consistent withhe proposed glycosylase-mediated generation of an abasic siten one of the strands, the repair intermediates could be digestedy the enzyme apurinic endonuclease (APE) [103–105]. Further-ore, introduction of 2′-deoxy-2′-fluoroarabinofuranosyl thymine

FdT) residues at the cross-link site shunted repair of the cross-ink to the incision-dependent (Fanconi anemia) pathway shown inig. 1. This result strongly implicated glycosylase activity in repairf the psoralen cross-link because the electron-withdrawing effectf the 2′-fluoro substituent in FdT (Fig. 2C) inhibits the action of BERlycosylases through destabilization of the oxocarbenium ion-likeransition state of these enzymatic reactions [81,82,106,107].

Repair of an Ap-derived cross-link was also investigated by Wal-

er and coworkers [94]. Ap sites are ubiquitous endogenous lesionsn cellular DNA [19,103,108] and recent work has characterizednterstrand cross-links arising from the reaction of the Ap aldehyde

ir 52 (2017) 1–11

residue with the exocyclic amino groups of adenine and guanineresidues in duplex DNA (Scheme 1) [17,109,110]. The Ap-derivedcross-links are chemically stable in duplex DNA [110,111] and thedA-Ap cross-link was previously shown to block DNA replication bythe strand-displacing polymerase �29, stalling primer extensionat the −1 position immediately preceding the cross-link [112]. Thestability of the Ap-derived cross-links may derive from the fact thatthey exist as cyclic aminoglycosides rather than in the ring-openedimine form (Scheme 1) [111,113,114]. The ubiquitous nature ofAp sites in genomic DNA makes endogenous cellular formation ofAp-derived cross-links an intriguing possibility.

In the Xenopus system, approximately 20% of the dA-Apcross-link was repaired by the FANCD2/FANCI incision-dependentpathway, while 80% of the repair proceeded via the incision-independent route [94]. As in the case of the psoralen cross-linkdescribed above, the repair intermediates could be digested by APE.Depletion of Rev1 prevented approximately half of the glycosylase-dependent repair, consistent with unhooking of the non-nativeglycosylase linkage in the cross-link to generate an Ap site onone strand and a native adenine residue on the other (Fig. 5 andScheme 1).

5. The glycosylase NEIL3 is involved in unhooking psoralenand Ap-derived interstrand cross-links

Semlow et al. provided evidence that the glycosylase NEIL3is responsible for unhooking of the psoralen and Ap-derivedcross-links in the Xenopus egg extracts [94]. For example, immun-odepletion of NEIL3 led to a decrease in repaired products andaddition of exogenous active NEIL3 enzyme to the assay reversedthis effect [94].

NEIL3 has a number of properties that are consistent with itsnewly defined role in replication-dependent cross-link repair. Theenzyme is expressed in early S and G2 phases of the cell cycle[115,116], during DNA replication and unpublished data cited inthe Semlow publication [94] notes that NEIL3 is associated with thereplisome. As discussed above, BER enzymes typically act on DNAsubstrate conformations in which the target base is extruded fromthe double helix (Fig. 2B) [81–83]. Clearly, a nucleobase involvedin a cross-link cannot be extruded in the typical manner. However,DNA structure at a stalled replication fork is atypical [48]. WhenNEIL3 unhooks cross-links at a stalled replication fork, the enzymepresumably must act upon a nucleobase that is located near thejunction of a strand-separated Y-shaped or X-shaped DNA struc-ture [2,3,34–36,48]. Significantly, NEIL3 displays preferences forthe removal of damaged bases from non-duplex substrates includ-ing single-stranded DNA, bubble structures, and G-quadruplexes[115,117,118]. There are a few examples of glycosylases that oper-ate without extrusion of the damaged base [119–122] and it willbe interesting to learn how the catalytic machinery of NEIL3 gainsaccess to the glycosidic bond of cross-linked nucleobases in thenon-canonical DNA structures generated at a stalled replicationfork.

It is possible to rationalize the observation that NEIL3 hasthe catalytic power to deglycosylate nucleobases involved in thepsoralen and Ap-derived cross-links [94]. To understand theseactivities it may be useful to compare the cross-link structures topreviously characterized NEIL3 substrates such as FapyA, FapyG,thymine glycol, and dihydrothymine [115,118]. The non-nativeglycosidic bond in the dA-Ap cross-link resembles the glycosidicbonds in the NEIL3 substrates FapyA and FapyG, while the satu-

substrates thymine glycol and dihydrothymine (Fig. 6). A previ-ous observation that the related BER glycosylase NEIL1 removespsoralen monoadducts from DNA provides further support for the

Z. Yang et al. / DNA Repair 52 (2017) 1–11 5

Scheme 1. Formation and structure of the dA-Ap interstrand cross-link.

Fig. 4. Formation and structure of a psoralen-derived interstrand cross-link. Panel A: Two sequential photoinduced [2 + 2] cycloaddition reactions generate interstrandc n fora arbonm

it

6i

iitiglec

ross-links preferentially at 5′-TA sequences in duplex DNA. The reaction is show psoralen-derived cross-link in duplex DNA. Thymine residues are shown with codification of pdb 204d).

dea that NEIL3 has the catalytic power to deglycosylate a psoralen-hymine adduct [93].

. Comparison of incision-dependent andncision-independent cross-link repair pathways

The evidence presented by Semlow et al. suggests that thencision-independent, glycosylase unhooking pathway involv-ng NEIL3 represents the front-line mechanism for repair ofhe two cross-links studied in the Xenopus system [94]. If thencision-independent pathway is thwarted, for example when

lycosylase-resistant FdT sugars are present at the psoralen cross-ink site, the slower incision-dependent (Fanconi) pathway isngaged (in general, these repair processes take place over theourse of several hours) [94].

the psoralent derivative trioxsalen. Panel B: The three-dimensional structure ofs colored yellow and psoralen with carbons colored in gray (image prepared by

It may be advantageous to unhook cross-links at a stalledreplication fork via an incision-independent pathway. In theincision-independent glycosylase pathway, the bifunctional (lyase)property of NEIL3 [115] is evidently suppressed, thus avoidingdouble-strand break formation (Figs. 3 and 5) [94]. In contrast,the actions of structure-specific endonucleases [45,47] in theincision-dependent Fanconi pathway generate a double-strandbreak (Fig. 1). Avoidance of double-strand break formation in theglycosylase-dependent unhooking mechanism eliminates the needto engage homologous recombination, a process that is quite com-plex in its own right and can lead to insertions and deletions

in the genome [69,123]. Both the incision-dependent (Fanconi)and incision-independent cross-link repair pathways include error-prone, potentially mutagenic steps [64,65,124], involving the

6 Z. Yang et al. / DNA Repa

Fig. 5. Model for replication-dependent, incision-independent repair of an Ap-derived interstrand cross-link. (a) NEIL3 unhooking of the cross-link by cleavageof the non-native glycosidic bond. (b) TLS and extension past the Ap site and normalextension past the native adenine residue. Repair of the Ap-containing duplex maybe completed by the BER proteins APE, pol �, and DNA ligase.

Fk

b[

ioregbNcna

ig. 6. Psoralen- and Ap-derived cross-links (left side) are structurally analogous tonown NEIL3 substrates (right side).

ypass of psoralen adduct remnants [62,63,125,126] and/or Ap sites127,128].

It may be important to mesh the new evidence implicating NEIL3n cross-link unhooking with literature supporting the involvementf Fanconi proteins, XPF/ERCC1, and double-strand breaks in theepair of psoralen-derived cross-links [129–131]. Three possiblexplanations were offered in this regard [94]: i. in some clono-enic survival experiments, the number of cross-links generatedy the psoralen-UV treatment may overwhelm the capacity of

EIL3, necessitating engagement of the incision-dependent Fan-oni pathway, ii. some cross-links generated in chromatin mayot be sterically accessible to NEIL3, thus requiring recognitionnd repair involving Fanconi proteins, iii. Fanconi proteins such as

ir 52 (2017) 1–11

FANCD2 may have critical functions in cross-link repair other thanunhooking. For example, FANCD2 can modulate chromatin dynam-ics [132] and may interact with FANCM in the reversal of stalledreplication forks [133].

It is also interesting to note that Neil3−/− knockout mice havebeen generated [134,135]. These mice do not display a profoundphenotype, although loss of proliferating neuronal progenitors wasnoted after hypoxia-ischemia, leading to the suggestion that “NEIL3exercises a highly specialized function through accurate molecu-lar repair of DNA in rapidly proliferating cells” [134]. The lack ofa severe phenotype in Neil3−/− mice, such as would be expectedto arise from cross-link repair deficiencies (e.g. Fanconi anemia)[41,44,45,136,137], could be due to the strongly overlapping func-tions of the Fanconi anemia and NEIL3-dependent repair pathways[94]. In addition, it is possible that overlapping functions of NEIL3and the related NEIL1 glycosylase in cross-link unhooking couldblunt the effects of NEIL3 deletion. NEIL1 displays substrate speci-ficity similar to NEIL3 in terms of the damaged nucleobases that itexcises and, like NEIL3, displays the ability to deglycosylate dam-aged bases located in non-duplex structures (in bubbles and nearnicks) [138]. Interestingly, NEIL1 is associated with the replisomeand a “cowcatcher” role has been proposed, in which the enzymecarries out pre-replication removal of oxidatively-induced lesionsfrom single-stranded regions of DNA at the replication fork [139].

7. New possibilities suggested by the role of NEIL3 incross-link repair: considering other enzyme activities thatcould catalyze replication-dependent unhooking ofinterstrand cross-links

The participation of NEIL3 in cross-link unhooking offers thepossibility of an entirely new role for BER glycosylases and otherDNA-processing enzymes. There are several properties that mightfavor the participation of any given enzyme in cross-link unhook-ing. i. Physical association with the replisome or Fanconi anemiaproteins such as activated FANCD2/FANCI could direct enzymeactivity to a cross-linked nucleotide located at a stalled replicationfork. ii. Unhooking enzymes must have catalytic activity on a cross-linked nucleotide located at the junction of Y-shaped, X-shaped,or Holliday junction (chickenfoot) structures at a stalled replica-tion fork [2,3,34–36,48,140]. In the case of NEIL3, such activitywas foreshadowed by studies demonstrating its ability to removedamaged nucleobases from non-canonical substrates such assingle-stranded DNA, quadruplex DNA, and bubbles [115,117,118].Borrowing terminology used to describe endonucleases such asXPF-ERCC1 [45,47], we could say that an enzyme such as NEIL3 has“structure-specific glycosylase” activity. iii. An enzyme involved inincision-independent unhooking of a cross-link must have the cat-alytic power to cleave one of the covalent bonds involved in theinterstrand cross-link. Although the mechanisms by which NEIL3gains access to the glycosidic bonds in the psoralen-derived anddA-Ap cross-links remain unknown, precedents (Fig. 6 and discus-sion above) indicate that the enzyme possesses the fundamentalchemical power to cleave the glycosidic bonds in these cross-links.

Each BER glycosylase has the catalytic power to process a lim-ited structural group of nucleobases [74–80]. There are manystructurally diverse cross-links [8,20] and the reactivity of eachparticular cross-link will define its susceptibility toward enzy-matic unhooking. In this regard, it is important to recognize thatinterstrand cross-links are not a generic, interchangeable, or homo-geneous group. Differences in the chemical structure and reactivity

of various cross-links are important. It is almost certain that manycross-links will be chemically resistant to the unhooking action ofNEIL3. However, other glycosylases may have the catalytic power tounhook cross-links that are refractory to NEIL3. For example, AAG

Z. Yang et al. / DNA Repair 52 (2017) 1–11 7

nt un

m[hm

Fig. 7. Speculative mechanisms for incision-independe

ay act upon cross-links derived from nitrogen mustards (Fig. 7A)141–143]. This is suggested by the ability of AAG to catalyzeydrolysis of the glycosidic bonds in N7-alkyl-dG and N3-alkyl-dAonoadducts [144]. Similarly, AAG has the chemical potential to

hooking of structurally diverse interstrand cross-links.

unhook the N1-dG attachment in a cross-link derived from the anti-cancer drug 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU, Fig. 7B)[145,146], as suggested by the ability of the enzyme to deglyco-sylate N1-methyl-dG [144]. AAG typically operates on extruded

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ucleobases [81,147] and it is completely unknown whether thective site of this enzyme can accommodate (and act upon) aross-linked nucleotide at a stalled replication fork. In this context,owever, it may be noteworthy that AAG is able to remove lesions

ocated in non-duplex (i.e. single-stranded) substrates [144].Some cross-links may not be amenable to unhooking by

lycosylase enzymes. For example, there are no BER enzymeshat deglycosylate platinated nucleobases generated by the anti-umor agent cisplatin [74–80]. Similarly, there are no knownER glycosylases that act on N6-alkyladenine, N2-alkylguanine,r N3-cytosine residues [74–80]. However, other classes ofNA-processing enzymes conceivably could unhook cross-linksontaining these types of chemical attachments. For example,he iron(II) and �-ketoglutarate-dependent dioxygenase activityf ALKBH family enzymes can carry out the oxidative dealkyla-ion of N3-alkylcytosine residues [148,149]. This chemistry couldnable unhooking of the N1-dG-ethyl-N3-dC cross-links derivedrom BCNU (Fig. 7C). Some isoforms of human ALKB enzymes actn non-duplex substrates [148,149].

The dioxygenase activity of ALKBH enzymes also brings theotential to unhook formaldehyde-derived [21] N6-dA-CH2-N6-A, N2-dG-CH2-N2-dG, N2-dG-CH2-N4-dC, and N6-dA-CH2-N4-dCross-links (Fig. 7E). ALKBH family enzymes are known to catalyzehe oxidative dealkylation of N6-alkyl-dA and N2-alkyl-dG residues148,149].

Enzymes with adenosine deaminase activity have the potentialo unhook N6-dA-CH2-N6-dA cross-links derived from formalde-yde (Fig. 7D) [21]. However, it must be noted that the enzymedenosine deaminase (ADA) has weak activity on oligomeric DNAubstrates [150]. ADAR enzymes are RNA editing enzymes thateaminate adenine residues in oligomeric RNA substrates [151].

nterestingly, Beal’s group showed that ADAR-2 can deaminate a 2′-eoxyadenosine residue located at the 5′-end of an RNA substrate152].

The deaminase activity associated with activation-induced cyti-ine deaminases (AID/APOBEC family of proteins) [153–155] hashe potential to unhook N4-dC-CH2-N6-dA cross-links derived fromormaldehyde [21]. Some RNA-editing enzymes also have theotential to catalyze deamination of cytosine residues in DNA [156]nd might participate in the unhooking of N4-dC-CH2-N6-dA cross-inks if this activity could be selectively directed at a cross-linkedytosine residue.

These selected examples (perhaps unnecessarily limited toonsideration of enzymatic activities associated with known DNA-rocessing enzymes) serves to illustrate how various enzymaticctivities could carry out the unhooking of structurally diversenterstrand cross-links. However, we emphasize that the aboveiscussion is highly speculative because the postulated “structure-pecific” activities deviate, substantially in some cases, from thestablished substrate specificities of these enzymes [157].

. Summary

Deep appreciation of various DNA repair pathways has beenuilt upon the in vitro characterization of the fundamental chem-

cal, biochemical, and structural properties of individual repairroteins [158–161]. Along these lines, it will be important to elu-idate the biochemical activities of NEIL3 (and other enzymes) onross-linked DNA substrates that resemble a stalled replication forkr transcription bubble. In addition, NEIL3 presents an excellentpportunity to elucidate the structural mechanisms by which a BER

nzyme can access the glycosidic bonds in a cross-linked duplex.n addition, chemical biologists can help address the roles of otherER glycosylases and ALKBH family enzymes in the replication-

ndependent unhooking of interstrand cross-links. For instance,

ir 52 (2017) 1–11

methods are in place for the preparation of duplex DNA substrateshousing a site-specific and structurally defined interstrand cross-link [17,110,146,162–174]. This will enable shuttle vector-basedmethods, in conjunction with genetic manipulation, for interrogat-ing the roles of BER glycosylases and ALKBH family proteins in theremoval of interstrand cross-link lesions in cells [164,175]. Like-wise, genetic modulation of these DNA repair enzymes, togetherwith measurements of interstrand cross-links in cellular DNA byliquid chromatography-tandem mass spectrometry (LC–MS/MS)[72,176–181], also offers the opportunity for exploring the rolesin these enzymes in the unhooking and repair of interstrand cross-links. At the same time, it will be important for studies in cellularsystems (and cell extracts) to establish how proteins present at astalled replication fork choreograph the action of NEIL3 and otherenzymes involved in cross-link unhooking, and to gather data fromliving organisms that shed light on the biological roles and potentialmedicinal relevance of these repair pathways.

Conflict of interest

The authors declare no conflict of interest

Acknowledgements

We apologize to authors of many important papers in the fieldthat were not cited due to space limitations. NP was supported inpart by an NRSA T32 institutional training grant (ES018827). KSGand YW are grateful to the National Institutes of Health for supportduring the writing of this review (ES 021007). We thank ProfessorOrlando Schärer for insightful comments on the manuscript.

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