Chemical approaches toward understanding base excision DNA repair

526

Chemical approaches toward understanding repair Orlando Despite

the

genome,

D Schker”,

importance

of DNA

the molecular

repair remains

poorly

repair pathway excise damaged on the recent

repair

In the base excision

of chemical

to the study

distinctive

of noncleavable

classes

complexes

of BER

recognize

involving

analyses

DNA glycosylases,

recently

have been

of protein-DNA

and for the isolation

An approach

DNA glycosylase-intermediate

complex

the mechanism

that

have

These analogs

and structural

that Several

analogs

glycosylases

of a novel DNA glycosylase. elucidate

and

approaches enzymes.

substrate

with DNA

and synthesized.

used for biochemical complexes

and

bases from DNA. This review focuses

development

been designed

recognition

DNA glycosylases

have been applied form stable

and Gregory

the

in protecting

basis for damage understood.

(BER),

Li Dengt*

to trap covalently

a

has also been used to

of DNA glycosylases.

Addresses *Department

of Cell Biology

and Genetics,

Erasmus

University,

PO Box 1738, 3000 DR Rotterdam, The Netherlands e-mail: [email protected] +Department of Chemistry and Chemical Biology, Harvard Cambridge, Massachusetts, 02138, USA

University,

ie-mail: [email protected] #e-mail: [email protected] Correspondence: Gregory L Verdine Current

Opinion

in Chemical

Biology

1997, 1:526-531

http://biomednet.com/elecref/1367593100100526 0 Current

Biology

Ltd ISSN

1367-5931

Abbreviations AP abasic BER base-excision repair pathway UDG uracil DNA glycosylase

base excision DNA

L Verdinel#

A major challenge in DNA repair research is to identify how BER enzymes recognize substrates that differ only subtly from their native counterparts, which are present in vast excess. Progress on this front ultimately requires high resolution structural and biochemical studies on protein-DNA complexes to elucidate the interaction between enzyme and substrate at the molecular level. Efforts along these lines have been hampered, however, by the necessarily fleeting nature of the association between these repair enzymes and their substrates. In principle, this problem could be overcome by modifying either the enzyme or the substrate co abolish catalysis while retaining substrate recognition, thereby generating a long-lived protein-DNA complex. Indeed, mutant versions of two DNA glycosylases, T4 endonuclease V and human uracil DNA glycosylase (UDG), have been found to form stable complexes with a substrate thymine dimer (a DNA modification caused by exposure to UV light) and the products of the UDG-cataluzed reaction, an AP site and free uracil, respectively [4,5,6”]. X-ray crystallographic studies of these complexes have provided insight into mechanisms of substrate specificity and catalysis of DNA glycosylases. This review focuses on an alternative approach that has emerged over the past few years, which relies on the modification of DNA substrates co obtain stable protein-DNA complexes involving BER enzymes. Several distinctive classes of uncleavable DNA analogs that interact specifically with DNA glycosylases have been designed and routes for their synthesis have been developed. These molecules have been used for biochemical and structural studies as well as for the isolation of a previously elusive DNA glycosylase.

Designed inhibitors Binding

Introduction Various

DNA repair pathways

exist in all known

organisms

to protect the genome from attacks by endogenous and exogenous agents [l]. Defects in DNA repair pathways are associated with a predisposition to cancer and affect cell viability [Z]. One of the major DNA repair pathways, the base excision repair pathway (BER), relies on DNA glycosylases to recognize aberrant base residues in DNA and to excise them through cleavage of the N-glycosidic bond linking the base to its deoxyribose sugar (see Figure 1) [3]. Two classes of DNA glycosylases are known: monofunctional DNA glycosylases which, generate abasic (AP) sites as products; and bifunctional DNA glycosylases/lyases, which can further catalyze the cleavage of the 3’ C-O bond through a p-elimination mechanism. Some DNA AP lyases are also able to mediate the cleavage of the 5’ C-O bond.

of DNA glycosylases

of DNA glycosylases

to stable

abasic

site

mimics

One of the first ‘synthetic’ DNA glycosylase inhibitors studies was the acyclic reduced abasic site (rAB, 1, Figure 2a) incorporated into a duplex oligonucleotide [7,8]. This abasic site mimic binds to a number of DNA glycosylase/AP lyases, but cannot be processed by these enzymes, because 1 cannot form the imine intermediate necessary for the p-elimination of the 3’ C-O bond [9]. The related tetrahydrofuran moiety (THF, 2, Figure 2a) [lo] has also been found to bind to various monofunccional and bifunctional DNA glycosylases. The mechanism

of DNA glycosylases

More recently, several classes of inhibitors have been designed according to the mechanism of the glycosidic bond hydrolysis reaction. The model for the reaction mechanism of DNA glycosylases is based on studies of enzymes such as glycosidases [l l] or nucleoside hydrolases [ 121

Chemical approaches toward understanding base excision DNA repair Schlrer,

527

Deng and Verdine

Figure 1

(basel

Damagedbase DNA glycosylase * DNA-O

DNA-O

DNA-O

DNA-O

Damagedbase site

Normalsite

X

Abasic site

Excision / replacement DNA synthesis

t

Current Opinion in Ctwmwi

The base excision

repair pathway.

Exposure

to various

DNA damaging

agents

results

in modified

bases

in DNA. DNA glycosylases

Bdcgy

recognize

the damaged bases and remove them from DNA by catalyzing scission of the N-glycosidic bond, monofunctional DNA glycosylases employ an activated water molecule as the nucleophile in the reaction, thereby generating an AP site as product (X=OH). DNA glycosylasesllysases emply an amine nuclephile on the enzyme and generate an aminal intermediate (X =NH-ENZYME), which undergoes further reactions (see Figure 4). In either case, the product of the glycosylase-catalysed reaction(s) is further processed by downstream enzymes in the BER pathway.

Figure 2

(a) 1’

OH

DNA-6

DNA-6

(1)

(2)

Reduced abasic site (rAB)

Tetrahydrofuran (THF)

DNA-O Proposed transition state structure

(5) Pyrrolidine homonucleoside

Pyrrolidine (PYR)

Current Opinion in Chemcal Biology

Abasic

site analogs

and transition

(2) have been used as stable transition molecule

state mimics

abasic

site mimics

as inhibitors

of DNA glycosylases.

and have been shown

(a) A reduced

to bind to a number

abasic

site (1) and a tetrahydrofuran

of DNA glycosylases.

(b) Structure

residue

of the proposed

state (3) of glycosidic bond cleavage catalyzed by DNA glycosylases. The nucleophile in this reaction is an enzyme-activated water in monofunctional DNA glycosylases, and an amine group from the enzyme itself in DNA glycosylases/AP lyases. The pyrrolidine (4)

was designed

to mimic the positive

charge

of the transition

state. The homopyrrolidine

which catalyze formally similar reactions. According to this model, the transition state is likely to resemble 3 (Figure 2b), in which substantial positive charge accumulates on the deoxyribose sugar ring, particularly at C-l’and O-l’. This model applies for both monofunctional and

nucleoside

5 is a base-containing

transition

state mimic.

bifunctional DNA glycosylases; although the nucleophile (Nu) in 3 is different. Monofunctional enzymes utilize an enzyme-activated water molecule, whereas the latter utilize an amino group on the enzyme itself as the nucleophile [9,13,14’]. Based on the proposed transition

528

Biopolymers

state structures,

glycosylase,

have state

two approaches for the design of inhibitors been pursued, involving the concepts of transition mimicry and transition state destabilization.

subnanomolar a 2’-hydrogen

Inhibitors

based

on transition

state

mimicry

The design of transition state mimics as inhibitors for DNA glycosylases was inspired by inhibitors of glycosidases [l l] and nucleoside hydrolase (12,151 which have a positively charged nitrogen in place of the endocyclic oxygen of the sugar moiety. A pyrrolidine analog of an abasic site (PYR, 4; Figure 2b) was incorporated into DNA to mimic the positive charge of the proposed transition state structure (3; Figure 2b) of the reaction catalyzed by DNA glycosylases [16]. Oligonucleotides containing a PYR residue were found to bind to a wide variety of monofunctional and bifunctional DNA glycosylases with subnanomolar affinity ([16,17] and SchErer OD, Nash HM, Jiricny J, Lava1 J and Verdine GL, submitted for publication). The role of the positively charged ammonium group in specific binding to DNA glycosylases was assessed by comparing the binding of PYR-containing oligonucleotides to that of the uncharged THF-containing oligonucleotides. Interestingly, in some cases the positive charge was of crucial importance for specific binding, whereas in many other cases the uncharged 2 and the charged 4 bound the enzymes with similar affinities. In the case of the DNA glycosylase AlkA, site-directed mutagenesis of Asp238-+Asn revealed that this residue is critically involved in a specific interaction with the ammonium group of the pyrrolidine [18’]. It was found that the Asp238-+Asn mutant lacks catalytic activity, indicating that the pyrrolidine moiety of the inhibitor binds in the active site of the enzyme [18*]. Although oligonucleotides containing the pyrrolidine abasic analog bind DNA glycosylases with high affinity, they are not suitable for studies aiming to elucidate the nature of base recognition. A new class of pyrrolidine-based inhibitors containing an attached base has recently been designed and synthesized to address the question of base recognition. To avoid stability problems with an aminal linkage, a methylene group was inserted between C-l’ and the base. The first example of this class of inhibitors, an adenine pyrrolidine homonucleoside (5; Figure 2b), exhibited subpicomolar affinity for the DNA glycosylase MutY [19”]. Inhibitors

based

on transition

state

destabilization

Anocher approach for designing inhibitors of DNA glycosylases has revolved around the concept of transition state destabilization. In the first examples of such inhibitors a fluorine atom was introduced at the 2’ position of the deoxyribose moiety of the substrate [20, 210,221. The electron withdrawing effect of the fluorine atom destabilizes the positive charge in the transition state (3); Figure 2b. Fluoronucleotides (6, 7; Figure 3) containing l,N6-ethenoadenine or uracil were not processed by the mammalian 3-methyladenine DNA glycosylase and G/T

respectively,

but bound

the

enzymes

in the

range [20,21’]. Interestingly, replacement by flourine in a thymine dimer substrate

of did

not prevent the ,v-glycosidic bond hydrolysis catalyzed by T4 endonuclease V, but did abolish the lyase reaction [22]. More recently, oligonucleotides containing deoxyribose versions of the natural products tubercidine (8; Figure 3) and formycin A (9; Figure 3) and aristeromycin (10; Figure 3) have been incorporated into DNA and shown to be resistant to cleavage by the MutY DNA glycosylase, while specifically binding the enzyme in the nanomolar to subnanomolar range [22,23-l. The resistance to enzymatic processing for these substrate analogs can be explained as follows: tubercidine lacks the N-7 nitrogen in the adenine base, which may be required for protonation by MucY to be activated as a leaving group; formycin A contains a C-glycosidic instead of an N-glycosidic linkage, which turns the base into a much poorer leaving group; and the carbocyclic aristeromycin lacks the free electron pairs bf the O-l’, which stabilizes the charge ordinarily developed on C-l’in the trabsition state. The use of inhibitors

in the study of DNA repair enzymes.

Complexes of DNA glycosylases with inhibitors have been analyzed using various biochemical methods. Nuclear resistance (footprinting) analysis of complexes of endonuclease III [9], MutY [23’,24], bacterial 8-oxoguanine DNA glycosylase (Fpg, Mutm) (HM Nash, GL Verdine, unpublished data) and the G/T glycosylase [Zl’] have been carried out to map the binding interaction between the enzymes and DNA. With the exception of the G/T glycosylase, all these enzymes protected an area of less than ten nucleotides on either DNA strand, indicating a small protein-DNA binding interface. The stretch of DNA bound by the G/T glycosylase is approximately twice as long,perhaps because the enzyme is much longer. hslethylation interference footprinting experiments of G/T glycosylase bound to an inhibitor revealed a specific interaction between the enzyme and a guanine residue in DNA that explains the observed sequence preference of the enzyme for G/T mismatches that occur in CpG sequences (the exclusive sites of cytosine methylation in mammals) [21*]. The mechanism of damaged base recognition by T4 endonuclease V is unusual in that it is the base opposite the thymine dimer (TT) lesion rather than the damaged base which is everted from the helix to adopt an extrahelical position. The combined use of pyrrolidine or reduced abasic site residues at the position of the substrate TT dimer as well as a fluorescent adenine analog 2-aminopurine have allowed the base-extrusion process to be observed using fluorescence spectroscopy

[251. X ray crystallographic studies of pyrrolidine-based inhibitors bound to two DNA glycosylases, AlkA and ANPG, have been initiated. Diffraction quality crystals have been obtained, and the structures of the complexes are expected to provide critical insight into the mechanisms

Chemical

Figure

approaches

toward understanding

base excision

DNA repair Schlrer,

Deng and Verdine

529

3

DNA-O

DNA-O

DNA-O

DNA-6

(6)

CI)

NH,

yH2

J- \ -N

DNA-O

DNA-O DNA-O

DNA-O

(9)

(6) 2’-deoxy

tubercidin

DNA-O

2’-deoxy formycin

(10) 2’-deoxy

I

aristeromycin Current Opinion in Chemical

Inhibitors of DNA glycosylases based on transition state destabilization. The electron withdrawing effects of the fluorine in the destabilizes the positive charge in the transition state. Tubercidin analog 7 lacks the N-7 nitrogen which is probably protonated glycosylase MutY to activate the base as a leaving group. Formycin A analog 6 contains a C-glycosidic linkage which is more enzymatic hydrolysis than the corresponding N-glycosidic linkage. The carbocyclic aristeromycin analog 9 lacks the endocyclic stabilizes the positive charge in the transition state.

of catalysis and DNA binding by these enzymes TE Ellenberger, personal communication).

(A Lau,

Inhibitors of DNA glycosylases are not only useful for the study of the nature of protein-DNA complexes. Recently, a yeast DNA glycosylase specific for &oxoguanine, yOgg1, was isolated using affinity chromatography using an duplex oligonucleotide containing a reduced abasic site as an affinity matrix [26]. This particular DNA glycosylase had previously eluded efforts at homology-based cloning due to the lack of sequence similarity with the corresponding bacterial DNA glycosylase. yOgg1 has also been independently isolated using complementation of a bacterial strain sensitive to damaging agents that generate S-oxoguanine lesions in DNA [27”]. Such a genetic screen may not be available for every lesion encountered by a DNA glycosylase, thus affinity purification may prove to be a powerful approach for the isolation of additional novel eukarytotic DNA glycosylases.

Covalent trapping of intermediates glycosylase/AP lyases

of DNA

Besides the design and synthesis of inhibitors of DNA glycosylases, another chemical assay has proven to be a valuable tool in the study of these enzymes. DNA

Bnlogy

2’ position of 6 by the DNA stable towards oxygen that

glycosylase lyases use an amine group on the enzyme to displace the damaged base at C-l’ (Figure 4) [9,13,14*,26,28]. Subsequent ring opening leads to an imino intermediate that renders the C-Z’protons more acidic and allows p-elimination of the 3’-phosphate group. The imino intermediate can be covalently trapped by reduction of the imine to the corresponding amine with sodium borohydride. This trapping assay has been used to determine whether or not DNA glycosylases contain an associated AP lyase activity and to identify the amino group of the enzyme that is responsible for the formation of the imino intermediate [13,14*,29]. This assay has also been very effective in following the enzymatic activity of DNA glycosylases during their purification from cell extracts [26**,30*,31*].

Conclusions A series of DNA glycosylase inhibitors have been recently designed using the concepts of transition state mimicry and transition state destabilization. The strong binding affinities demonstrated for several of these inhibitors have validated the transition state structure model on which their design was based. The approaches used to design inhibitors are general in nature and are, therefore, applicable in principle to any DNA glycosylase. Structural

530

Biopolymers

Figure

4

IMlon, 11

O’deoxyrlbow, 16

covalent rdduct. 12

7’

s’-DNA-OH

HykEnz R

reduced Schlff brsa, 17 a stable, covalenl adduct

Schlfl base, 13

” EL,,

Enz

15

14

Current Opinion in Chemical Biology

The trapping assay for DNA glycosylases/AP lyases. An amine nucleophile displaces the damaged base and forms a covalently linked enzyme-substrate aminal intermediate (11). Rearrangement of 11 generates imine 12. Enzyme assisted abstraction of the acidic 2’-H in 12 leads to enamine 13, which undergoes conjugate elimination to cleave the 3’C-0 bond. The a, P-unsaturated imine thus generated is further processed by AP lyases and additional enzymes in the BER pathway. Sodium borohydride can intercept the imine intermediate 12, resulting in the formation of an irreversible enzyme DNA cross-link 14.

studies of complexes of DNA glycosylases bound to inhibitors are still in their infancy but will provide much needed insight into the mechanism of damage recognition and catalysis by BER enzymes. Various DNA glycosylases counteract the damage done to the DNA of cancer cells by alkylating agents and ionizing radiation in cancer therapy. Inhibitors of DNA glycosylases might therefore be used in the future to enhance the effects of such therapeutic agents.

Acknowledgements

excision repair enzyme complexed with a DNA substrate: structural basis for damaged DNA recognition. Cell 1995, 831773-702. 6. ..

Slupphaug G, Mol CD, Kavil B, Arvai AS, Krokan HE, Tainer JA: A nucleotide-flipping mechanism from the structure of human uracil-DNA glycosylase bound to DNA. Nafure 1996, 80:87-92. This paper and the one by Vassylyev et al. (1995; 151) provide the first two crystal structures of DNA glycosylases bound specifically to their target DNAs. In both cases the mutation of active site residues of the enzymes led to the formation of a stable enzyme-substrate complex. An adenine base opposite the substrate base in the T4 endonuclease V complex, and the targeted uracil itself in the uracil DNA glycosylate both adopt an extrahelical position. 7.

Castaing B, Boiteux S, Zelwer C: DNA containing a chemically reduced apurinic site is a high affinity ligand for the E. co/i formamidopyrimidine-DNA glycosylase. Nucleic Acids Res 1992, 20:389-394.

8.

O’Handley S, Scholes CP, Cunningham RP: Endonuclease Ill interactions with DNA substrates. 1. Binding and footprinting studies with oligonucleotides containing a reduced apyrimidinic site. Biochemistry 1995, 34:2528-2536.

9.

Dodson ML, Michaels ML, Lloyd RS: Unified catalytic mechanism for DNA glycosylases. J Viol Chem 1994, 52:32709-32712.

10.

Takeshita M, Chang C-N, Johnson F, Will S, Grollman AP: Oligodeoxynucleotides containing synthetic abasic sites. J Biol Chem 1987, 262:10171-l 0179.

11.

McCarter JD, Withers SG: Mechanisms of enzymatic hydrolysis. Curr Opin Struct Viol 1994, 4:885-892.

12.

Schramm VL, Horenstein BA, Kline PC: Transition state analysis and inhibitor design for enzymatic reactions. J Biol Chem 1994, 269:18259-l 8262.

13.

Zharkov DO, Rieger RA, lden CR, Grollman AP: NHS-terminal proline acts as a nucleophile in the glycosylase/AP lyase reaction catalyzed by Escherichia coli formaminopyrimidineDNA glycosylase (Fpg) protein. J Viol Chem 1997, 272:53355341.

‘l-his work in the author’s lab on rhis subject \vas supported in part by National Instlture of Health grant 51330 (w Gregory 1, \trdinc). Li Deng is supported by a postdoctoral fellowship from the American Cancer Society.

References and recommended

reading

Papers of particular interest, published within the annual period of review, have been highlighted as: . l

*

of special interest of outstanding interest

1.

Lindahl T: Instability and decay of the primary Nature 1993, 362:709-715.

structure

2.

Friedberg EC, Walker GC, Siede W: DNA Repair and Mutagenesis. Washington, DC: ASM Press; 1995.

3.

Seeberg E, Eide L, Bjsr% M: The base excision Trends Biochem Sci 1995, 20:391-397.

4.

Vassylyev DG, Morikawa K: DNA-repair struct Biol 1997, 7:103-l 09.

5.

Vassylyev DG, Kashiwagi T, Mikami Y, Ariyoshi M, lwai S, Ohtsuka E, Morikawa K: Atomic model of a pyrimidine dimer

enzymes.

repair

of DNA.

pathway.

Curr Opin

glycoside

Chemical

approaches

toward

understanding

Nash HM, Lu R, Lane WS, Verdine: The critical active site amine of the human 8-oxoguanine DNA glycosylase, hOgg1: direct identification, ablation and chemical reconstitution. Chem Biol 1996, 4:693-702. This paper reports the identification of the active site amine of human 8oxoguanine DNA glycosylase (hOgg1). Edman sequencing of an active site peptide covalently linked to substrate DNA by reductive trapping of the imine intermediate directly identifies the E-HNZ group of Lys249 as the active imine. 14. .

15.

16.

1 7.

Look GC, Fotsch CH, Wong C-H: Enzyme-catalyzed organic synthesis: practical routes to aza sugars and their analogs for use as glycoprocessing inhibitors. Accounts Chem Res 1993, 28:182-l 90. Schlrer OD, Ortholand J-Y, Ganesan A, Ezaz-Nikpay K, Verdine GL: Specific binding of the DNA repair enzyme AlkA to ;6p;;ylidine-based inhibitor. I Am Chem Sot t 995, 117:6623McCullough AK, Scharer OD, Verdine GL, Lloyd RS: Structural determinants for specific recognition by T4 endonuclease V. J B/o/ Chem 1996, 271:32147-32152.

Labahn J, Scharer OD, Long A, Ezaz-Nikpay K, Verdine GL, Ellenberger TE: Structural basis for the excision repair of alkylation-damaged DNA. Cell 1996, 86:321-329. The cyrstal structure of the DNNA glycosylase Alka reveals a binding pocket for the alkylated bases recognized by the enzyme lined with electron rich aromatic amino acids. An aspartate residue (Asp238) that points into the binding pocket is shown to be essential for catalysis by site-directed mutagenesis. Mutation of Asp1 38 diminishes binding of a pyrrolidine inhibitor by 50 fold, indicating that the positively charged pyrrolidine ring interact with Asp238 in the active site. 18. .

Deng L, Schlrer OD, Verdine GL: Unusually strong binding of a designed transition state analog to a base-excision DNA repair protein. 1 Am Chem Sot 1997, II 9:7865-7866. This paper describes the synthesis of an oligonucleotide containing an pyrrolidine homoadenine residue as a transition state mimic of the DNA glycosylase MutY. This inhibitor binds to its target DNA glycosylase MutY more than an order of magnitude more tightly than any other DNA glycosylase inhibitor to date. 19. ..

20.

Schlrer OD, Verdine GL: A designed inhibitor of base-excision DNA repair. J Am Chem Sot 1995, 117:10781-10782.

21. ..

Scharer OD, Kawate T, Gallinari P, Jiricny J, Verdine GL: Investigation of the mechanisms of DNA binding of the human G/T glycosylase using designed inhibitors. Proc Nat/ Acad Sci USA 1997, 94:4878-4883. The concecpt of transition state destabilization for the inhibition of DAN glycosylases is developed in this paper. Introduction of a fluorine atom at C2’of the substrate nucleotide results in a substrate analog that is bound specifically by DNA glycosylase but no longer processed. Methylation interference analysis of an G/T glycosylase-inhibitor complex revealed a specific contact of the enzyme to a G residue adjacent to the G/T mismatch, which helps to explain the observed sequence preference of the enzyme. 22.

lwai S, Maeda M, Shirai M, Shimada Y, Osafune T, Murata T, Ohtsuka E: Reaction mechanism of T4 endonuclease V determined by analysis using modified oligonucleotide duplexes. Biochemistry 1995, 11:4601-4609.

23. .

Porello SL, Williams SD, Kuhn H, Michaels ML, David SS: Specific recognition of substrate analogs by the mismatch repair enzyme MutY. J Am Chem Sot 1996, 118:10684-l 0692.

base excision

DNA repair Schlrer,

Deng and Verdine

531

Oligonucleotides containing modified adenine bases derived from the natural products tubercidine and formycin A are bound specifically, but not processed, by the DNA glycosylase MutY. 24.

Bulychev NV, Varaprasad CV, Dorman G, Miller JH, Eisenberg M, Grollman AP, Johnson F: Substrate specificity of Escherichia co/i MutY protein. Biochemistry 1996, 35:13147-l 3156.

25.

McCullough AK, Dodson ML, Schlrer OD, Lloyd RS: The role of base flipping in damage recognition and catalysis by T4 endonuclease V. J Viol Chem, 1997, 232:27210-27217.

26.

Nash HM, Bruner SD, Scharer OD, Kawate T, Addona TA, Spooner E, Lane WS, Verdine GL: Cloning of a yeast 8oxoguanine DNA glycosylase reveals the existence of a baseexcision DNA repair protein superfamily. Gun Biol 1996, 6:966980.

27. ..

Van Der Kamp PA, Thomas D, Barbey R, de Olivera R, Boiteux S: Cloning and expression in fscherichia co/i of the OGGl gene of Saccharomyces cerevisiae, which codes for a DNA glycosylase that excises 7,8-dihydro-8-oxoguanine and 2,6diamino-4-hydroxy-5-N-methylformylamidopyrimidine. Proc Nat/ Acad Sci USA 1996, 93:5197-5202. This paper and that of Nash et al. [27] report the cloning and characterization of a yeast 6-oxoguanine DNA glycosylase. Nash et a/. [261 used the sodium borohydride trapping assay to reveal the presence of a E-oxoguanine DNA glycosyalse / lyase activity in yeast cell extracts. The protein was then isolated using affinity chromatography with an oligonucleotide containing a reduced absic site residue.Van der Kamp et a/. [27] isolated the same protein using a genetic screen by expressing a yeast cDNA library in an Ecoli strain deficient in 8-oxoguanine DNA glycosylase activity. The yeast 8-oxoguanine DNA glycosylase is not homologous to the corresponding bacterial enzyme and therefore coulld not be cloned based on homology alone. The two papers demonstrate that chemical and genetic approaches can be complementary in isolation and cloning of eukaryotic DNA repair enzymes. 26.

Sun B, Lathan KA, Dodson M, Lloyd RS: Studies on the catalytic mechanism of five DNA glycosylases. Probing for DNA enzyme imion intermediates. J Biol Chem 1995, 270:32709-32712.

29.

Dodson ML, Schrock RD, Lloyd RS: Evidence for an imino intermediate in the T4 endonuclease V reaction. Biochemisty 1993, 326264-8290.

30. .

Lu R, Nash HM, Verdine GL: A mammalian DNA repair enzyme that excises oxidatively damaged guanines maps to a locus frequently lost in lung cancer. Curr Biol 1997, 7:397-407. The use of borohydride trapping for the identification and characterization of 8-oxoguanine DNA glycosylase / lyase activities in human and nurine cells is described. The cloning of corresponding glycosylases identifies them as members of the superfamily of BER proteins. 31. .

Hilbert TP, Boorstein RJ, Kung HC, Bolton PH, Xing DH, Cunningham RP, Teebor GW: Purification of a mammalian homologue of Escherichia co/i endonuclease Ill: Identification of a bovine pyrimidine hydrate thymine glycol DNA glycosylase/AP lyase by irreversible cross linking to a thymine glycol-containing oligonucleotide. Biochemistry 1996, 35:25052511. The purification of a mammalian homolog of endonuclease Ill (a DNA glycosylase which acts to excise oxidised pyrimidines) is achieved by the ability of the protein to form a Schiff base intermediate with a radioactively labelled thymine glycol containing oligonucleotide that can be trapped with NaCNBHs. An enzymatic activity from calf thymus was purified over several chromatographic separations and isolated by monitoring its DNA glycosylase/AP lyase activity using the trapping assay.