Unified catalytic mechanism for DNA glycosylases.

THE JOURNAL OF B I ~ L ~ C ~ CC HA EM L IS~Y Vol. 269,No. 52,Issue of December 30,pp. 32709-32712, 1994 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

Minireview

mechanism). Although the characteristics of this enzyme have been reviewed, it is worthwhile highlighting a number of the contributions madeusing this enzyme as a prototype or model for the other DNA glycosylases and DNA glycosylasdAP lyases. M. L. DodsonS, Mark L. Michaelsl, and Endonuclease V was the firstDNA repair enzyme to have its R Stephen Lloyda structure determined by x-ray crystallography (11).The structure consisted of a three-helix bundle in a single domain with From the scaly Center for Molecular Science, the several amino acid residues at the N terminus wedged University of n x a s Medical Branch, Galveston, Texas 77555 and the U m g e n Corporation, between a-helices 1and 3. Specific amino acid residues, previThousand Oaks, California 91320 ously shown t o be critical for binding to nontargetDNA and for the i n vitro and i n vivo processivity of the enzyme, were clusThe structural integrity of the DNA bases within cells is tered around the N terminus in the structure. The final 12-14 continuously being challenged by both spontaneous decompo- C-terminal residuesof endonuclease V, containing multiple arsition anda variety of damaging agents suchas chemicals and omatic and basic residues, were previously implicated in pyUV and ionizing radiation. In response, cells have elaborate rimidine dimer (substrate) binding (12-16). This is of some mechanisms torectify DNA damage, including nucleotide exci- interest since this portion of the enzyme is located approxision (1-3) and recombinational (4-6) repair pathways. In ad- mately 15-20 from the active site in the structure. This dition to these, the cellular base excision repair system pro- seeming structural disparity may indicate that a conformavides a major line of defense against mutagenesis and cell tional change contributest o substrate binding. death (7). As mentioned above, endonuclease V binds to nontargetDNA DNA base excision repair canbe thought of as a sequence of in addition topyrimidine dimers. As reviewed by Lohman (17) steps beginning with therecognition of either a specific type of and von Hippel and Berg (181, this nontarget DNA affinity, damaged DNA structure or a n inappropriate base, e.g. uracil. which is manifested in many DNA interactive proteins (poThe many enzymes that can initiate the base excision repair lymerases, repressors, restrictionimodification enzymes, DNA pathway, the DNAglycosylases, do so by catalyzing theremoval repair enzymes), reduces the dimensionality of the diffusive of the inappropriatebase. These enzymes monitorDNA for the search within a cell to locate specific DNA sequence arrays or presence of damaged or inappropriate bases,bind to those sites, sites of DNA alterations. These interactions aregenerally elecand catalyze the breakageof the N-(2-1’ glycosyl bond. One of trostatic in nature, although hydrophobic contacts may also be the most obvious differences among these variousglycosylases important. Although endonuclease V is one of many proteins is that some catalyze the formation of a DNA single strand that have been shown to employ this one-dimensional scanning break at thenew AP’ site created by the initial glycosyl bond or looping on DNA for substrate binding i n vitro (19-22), it was of the glycosylase the first DNA repair enzyme for which this mechanism was scission at a rate that approximates the rate step, while for others, the rate of formation of single strand shown t o occur i n vivo (23). It was also the first protein for breaks is at least 2 orders of magnitude slower than theglyco- which this mechanism of target sitelocation was shown to be of sylase step. In all cases examined, the “endonuclease” activity biological significance (24-29). In the latter studies, it was of the DNA single strand breaking enzymes followed a lyase demonstrated that mutantenzymes that retainedfull catalytic type of chemistry rather thana phosphoryl transfer pathway. activity but hadlost their ability to processively scan nontarget In these cases, the break in the sugar-phosphate backbone had DNA were unable to enhance the survival of repair-deficient a 3’-a,P-unsaturated aldehyde and a 5”phosphate as termini Escherichia coli cells or T4 phage following UV irradiation. (8). After the release of the altered base and independentlyof Endonuclease V was also the first DNA glycosylase or DNA whether there is anassociated lyase activity, these sites must glycosylase/AP lyase to have its active site residue identified be processed by an additional endonuclease to create a 3‘-hy- (30). Althoughit hadbeen suggested previously that an amine droxyl group, thenecessary substrate for subsequent polymer- group could be responsible for catalyzing the lyase reaction by ization and ligation (7). a @elimination mechanism, chemical modification of the enMany differenttypes of DNAglycosylases or DNAglycosylase/ zyme established this point. Reductive methylation of amine AP lyases have been described. They are generally small pro- groups on the protein demonstrated that modification of the teins, ranging in molecular mass from 16 to42 kDa, anddo not a-amino group of the N-terminal threonine residue led to a require metalcofactors or exogenous energy sourcesfor activity. stoichiometric inactivation of both the DNA glycosylase and AP Except for uracil DNA glycosylase, all DNA glycosylases show a lyase activities without interfering withpyrimidine dimer-spestrong preference for incising bases from double-stranded ver- cific binding (30). The involvement of the N terminus was corsus single-stranded DNA. roborated further by site-directed mutagenesis (31).The idenOne of the most extensively studied of these enzymes is T4 tification of a n imino intermediate, a hallmark of this type of endonuclease V. It is a cyclobutane pyrimidine dimer-specific chemistry, was demonstrated recently (32). DNAglycosylasdAF’lyase, and its biochemical properties were exThese studies have led to the development of a complete tensively described and reviewed (see Ref. 9 for a s u m m a r y of the reaction scheme for endonuclease V. A generalized mechanism early literature and Ref. 10 for a recent overview of the catalytic for the chemistryof the combined DNA glycosylase and abasic site lyase activities is diagrammed schematically in Fig. 1, * This minireview will be reprinted in theMinireview Compendium, panels C-E. This is one exampleof a type of well known chemwhich will be available in December, 1994. 1 To whom correspondence should be addressed. Tel.: 409-772-2179; istry employed by enzymes for catalyzing the cleavage of the second C-C or C-H bond away from a carbonyl carbon. In their Fax: 409-772-1790. The abbreviation used is: AP, apurinic or apyrimidinic (site). textbooks Jencks and Fersht (33,34) have described the Schiff

Unified Catalytic Mechanism for DNA Glycosylases*

a

32709

Minireview: Unified Catalytic Mechanism for DNA Glycosylases

32710

n

B

D

trans a-8

unsaturated aldehyde

FIG.1. Proposed mechanism of DNA glycosylases. Panel A diagrams the proposed mechanisms for DNA glycosylases and DNA glycosylasdAP lyases.The parts of the figure in black depict reactions common to the two glycosylasetypes. Portions in blue are specific to those glycosylases without an accompanyingAP lyase, and red highlights the reactions specific to glycosylases with lyase activity. The two pathways differ in the kind of nucleophile attacking the C-1’ sugar carbon of the damaged nucleoside; glycosylase/APlyases employ a nucleophile on the enzyme, the N-terminal a-amino group in thecase of endonucleaseV, possibly lysine 120 in thecase of endonuclease111, while glycosylaseslacking AP lyase activity use a nucleophile fromthe medium, most likely hydroxide ion or an activated water molecule. Punel B illustrates the chemistry thought to occur in reaction B ofpanelA. The base, here shown as uracil, is likely protonated to make it a better leaving group, but no information is available to indicate where on the ring thismight occur. Panel C shows the chemistry of the glycosylase step of glycosylase/AP lyases. The base is illustrated as a thymine dimer, the substrate for endonucleaseV. Again, the base is shown beingprotonated but without indicating the site of this protonation. Punel D shows the chemistry of dissociation of the common intermediate for the glycosylasdAPlyase enzymes. This corresponds to the reverse of reaction D in panel A. Panel E illustrates the actual lyase step, beginning with protonated Schiff base intermediate. The stereochemicalcourse was determined be Gerlt and co-workers (8).

base formed in this chemistry as readily protonated, which converts it into an effective “electronsink.” Examples of the use of such Schiff base intermediates to activate C-C and C-H bonds cited in these textbooks include the aldolase mechanism and thecovalent catalysis of the decarboqlationof p-keto acids. This type of mechanism is characterized by several diagnostic experimental hallmarks: covalent trapping of the covalent enzyme-substrate complex by reduction with NaBH, and inhibition of the reaction by cyanide in the presence of substrate, for example. These hallmarks characterize the T4 endonuclease V reaction and constitute strong support for this mechanism (32). The key points of this scheme (Fig. 1, panels C-E) are as follows. 1) There are two different types of damage in DNA that serve as substrates for endonuclease V: ( a )sites with a denuded sugar moiety (these are also known as abasic or apurinic (AP) sites because they were first described as theproducts of acid depurination of DNA) and ( b )sites of cyclobutane pyrimidine dimers. 2)After formation of the appropriate Michaelis complex

(represented as E-damaged base site or E-abasic site in Fig. 1, panel A) both substrates are converted to a common type of structural intermediate. This intermediate consists of C-1’of the DNA covalently bound to the nitrogen of the N-terminal cy-amino group of the protein. 3) The fate of this covalent imino intermediate is to be hydrolyzed, either before (panel D )or after (panel E ) the DNA undergoes a p-elimination reaction resulting in scission of the phosphodiester backbone of the DNA (the abasic site lyase step). 4) Finally, the enzyme dissociates from the product site butremains associated with the nontarget DNA for a time dependent on the saltconcentration in the medium (19-21). Since the common intermediate can dissociate before the Felimination step (correspondingto the reverse reaction D of Fig. 1,panel A) or after the p-elimination step (reaction E ), the propensity of a DNA damage-specific glycosylase to catalyze a single strand break can be thought of as a competition between the rates of reactions D (reverse) and E . Panels D and E depict the chemistry thought tooccur in these two steps.

Minireview: Unified Catalytic Mechanism We propose a unifying hypothesis that rationalizes the apparent distinction betweenthe two types of base excision repair glycosylases: those which catalyze a DNA single strand break at a rate approximating thatof the glycosylase step and those with glycosylase activity only. This hypothesis is based on similarities and distinctions in catalytic mechanism between the two generalized types of enzymes and is diagrammed in Fig. 1, panel A. The black portions of the diagram indicate common the steps for the two types of mechanism. The blue arrows ( B ) describe the proposed reaction components specific for glycosylases without a n accompanying lyase activity. The red arrows indicate the reaction path specific to DNA glycosylases/AP lyases. The bifurcation between the two reaction types can be seen tolie with the typeof nucleophile that attacksC-1’ of the “damaged base” nucleoside. We propose that all DNA damagespecific glycosylases that catalyze a phosphodiester backbone scission at a rate approximately equal t o the rate of the glycosylase step go through an imino intermediate of the type described for the T4 endonuclease V reaction mechanism. Glycosylases lacking the ability catalyze to DNAsinglestrand breaks in this facile manner use a nucleophile derived from the medium (suchas a hydroxide ion or an activated watermolecule). Experimentally, the latter mechanism can be distinguished from the former because no enzyme-DNA covalent intermediate is involved. Is there data from studies of other DNA glycosylases/AP lyases that might support extending the endonuclease V mechanism in thisway? Recently such evidence has been found for the adenine-specific DNA glycosylase that is the product of the E. coli mutY gene. The mutY product is a member of a new family of a structurally related, 4Fe-4S cluster, DNA glycosylases/AP lyases of widely divergent substrate specificities. This group includes E. coli endonuclease 111, which initiates baseexcision repair on thymineglycol lesions (35, 36)as well as a number of other ring-saturated andring-fragmented derivatives of thymine, Micrococcus luteus UV endonuclease, which initiates repair of pyrimidine dimers (37), and MutY, which removes an undamaged adenine when it is mispaired opposite a n 8-oxo-guanine lesion, an 8-oxo-adenine lesion, guanine, or cytosine (3842). Although all three enzymes are glycosylases, only endonuclease I11 and UV endonuclease have AP lyase rates that are approximately equal to the rate of base excision. The rate of phosphodiester backbone cleavage for MutY is much slower than its glycosylase rate. The three-dimensional structure of E. coli endonuclease 111, the prototype member of the group, has been solved by x-ray crystallography (43, 44). Endonuclease I11 is composed of two domains approximately equal in size but separated by a deep cleft the width of one water molecule. One of the domains is composed of six antiparallel a-helices with nearest neighbor connectivity. The six helices are arranged such that one is centrally located within the remaining five helices. The second domain contains four a-helices, one from the N-terminal sequence and the remaining three coming from the C-terminal portion. The 4Fe-4S cluster is located completely within the C-terminal a-helices, with the coordination sequence motif beA thymine glycol-binding site ing Cys-X6-Cys-X2-Cys-X,-Cys. was identified as a @-hairpin structure near residues113-119 by difference Fourier mapsobtained after soaking crystals ina thymine glycol solution. The alignment of the sequences of MutY, endonuclease 111, and M. luteus UV endonuclease in a region near theendonuclease I11 thymine glycol binding sitesis shown in Fig. 2. Note that 10 residues in this sequence region are identicalexcept for the residuecorresponding to lysine 120 in the endonuclease 111sequence. MutY has a serine replacing the lysine of the other two enzymes.

for DNA Glycosylases End03 M.luteus W MutY

32711

110

120

130

I

I

I

GEVPEDRAALEALPGVGRKTANVVLNTAFGWPTI GEVPARLEDLVALPGVGRKTAFVVLGNAFGQPGI GKFPETFEEVAALPGVGRSTAGAILSLSLGKHFP

FIG.2. protein sequence alignmentfor some DNAglycosylase/AP lyases. Alignment of the sequencesofE. coli endonuclease111,M . lutezu W endonuclease,and the E. coli mutY gene product in the region of the endonuclease I11 “thymineglycol-binding site” is shown.The underlined amino acid residuesrepresent the near identitybetween the three proteins, with the only difference being the MutY serine at the position corresponding to the endonuclease I11 sequence. The sequence for M . luteus UV endonuclease should be considered preliminary.

Analysis of the crystal structure of endonuclease 111 led to the proposal that lysine 120 was the most likely candidate for the formation of the Schiff base associated with the AP lyase activity of endonuclease 111. SinceMutY lacks this critical amino acid one would predict that MutY would lack AP lyase activity. In fact, MutY was initially characterized as strictly a glycosylase (40,41). Others,however, have reported that an AP lyase activitycopurifies with MutY (45). In wild type MutY this “APlyase” rate is much slower than theglycosylase step. When serine 120 is changed to a lysine, however, the glycosylase and AP lyase ratesare approximatelyequal. By analogy, this strongly suggests that the €-amino group of lysine 120 is involved in the strandcleavage reaction of endonuclease I11 and M. luteus UV endonuclease. From the crystal structure of endonuclease 111, glutamate 112 was predicted to be involved in the glycosylase reaction. MutY does not have an acidic residue corresponding to that exact position, but it does have glutamatesat positions 109and 110. When these were mutated to glutamine either as single mutations or as the double mutation, the mutants retained wild type activity. Further analysis of the crystal structure of endonuclease I11 suggested that aspartate138, which is located at the end of a n a-helix in the 4Fe-4S domain opposite lysine 120 might be involved in catalysis. When this residue was changed from aspartate to asparagine, MutY lost glycosylase activity though it retained the ability to bind to its substrate. This evidence suggests that aspartate 138 is an active site residue in MutY. REFERENCES 1. Sancar, A,, and Tang, M. (1993) Photochem. Photobiol. 67, 905-921 2. Hoeijmakers, J. H. J. (1993) Dends Genet. 9, 173-177 3. Hoeijmakers, J. H. J. (1993) P e n d s Genet. 9,211-217 4. Kowalczykowski, S. C. (1991)Annu. Reu. Biophys. Biophys. Chem. 20,539-575 5. West, S. C. (1992)Annu. Reu. Biochem. 61,603440 6. West, S. C. (1993) in Nucleases (Linn, S. M., Lloyd, R. S., and Roberts, R. J., eds) Vol. 11, pp. 145-170, Cold Spring Harbor Laboratory Press, Plainview,

NY 7. Lloyd, R. S., and Linn, S. (1993) in Nucleuses (Linn, S. M., Lloyd, R. S., and Roberts, R. J., eds) Vol. 11, pp. 263-316, Cold Spring Harbor Laboratory Press, Plainview, NY 8. Gerlt, J. A. (1993) in Nucleases (Linn, S. M., Lloyd, R. S., and Roberts, R. J., eds) Vol. 11, pp. 1-334, Cold Spring Harbor LaboratoryPress,Plainview, NY 9. Dodson, M. L., and Lloyd, R. S. (1989) Mutat. Res. 218,49-65 10. Latham, K. A,, and Lloyd, R. S. (1994) Nucleic Acids Res.,in press 11. Morikawa, K., Matsumoto, O., Tsujimoto, M., Katayanagi, IC, Ariyoshi, M., Doi, T.,Ikehara, M., Inaoka, T., and Ohtsuka, R. (1992) Science 256, 523526 12. Stump, D. G., and Lloyd, R. S. (1988) Biochemistry 27, 1839-1843 13. Recinos, A,, and Lloyd, R. S. (1988) Biochemistry 27, 1832-1838 14. Lloyd, R. S., and Augustine, M.L. (1989) Proteins Struct. Funct. Genet. 6, m-138 15. Ishida, M., Kanamori, Y.,Hori, N.. Inaoka, T., andOhtsuka, E. (1990) Biochemistry 29,3817-3821 16. Latham, K. A., Carmical, J. R., and Lloyd, R. S . (1994) Biochemistry 33, 9024-9031 17. Lohman, T.M. (1986) Crit. Reu. Biochum. 19, 191-245 18. yon Hippel, P. H., and Berg, 0. G. (1989) J . Biol. Chem. 264, 675478 19. Lloyd, R. S.,Hanawalt, P. C., and Dodson. M. L. (1980) Nucleic Acids Res. 8, 5113-5127 20. Ganesan, A. K., Seawell, P., Lewis, R. J., andHanawalt, P. C. (1986) Biochemistry 26,5751-5755 21. Gruskin, E.A,, and Lloyd, R. S. (1986) J . B i d . Chem. 261,9607-9613

32712

Minireview: Unified Catalytic Mechanism for DNA Glycosylases

22. Lloyd, R. S., Dodson, M. L., Gruskin, E. A,, and Robberson, D. L. (1987)Mutat. Res. 183, 109-115 23. Gruskin, E.A,, and Lloyd, R. S. (1988)J. Biol. Chem. 263,12728-12737 24. Dowd, D. R., and Lloyd, R. S. (1989)J. Mol. Biol. 208, 701-707 25. Dowd, D. R., and Lloyd, R. S. (1990)J. Biol. Chem. 266,3424-3431 26. Nickell, C., and Lloyd, R. S. (1991)Biochemistry 30, 863M648 27. Nickell,C.,Anderson, W. F., and Lloyd, R. S. (1991)J. Biol.Chem. 266, 5634-5642 28. Nickell, C., Prince,M. A,, and Lloyd, R. S. (1992)Biochemistry 31,4189-4198 29. Augustine, M. L., Hamilton, R. W., Dodson, M. L., and Lloyd, R. S. (1991) Biochemistry 30,80524059 30. Schrock, R. D., 111, and Lloyd, R. S. (1991)J. Biol. Chem. 266, 17631-17639 31. Schrock, R. D., 111, and Lloyd, R. S. (1993)J. B i d . Chem. 268, 880-886 32. Dodson, M. L., Schrock, R. D., and Lloyd, R. S. (1993)Biochemistry 32,82848290 33. Jencks, W. P. (1987)i n Catalysis in Chemistry and Enzymology(Jencks, W. P., ed) pp. 111-150,Dover Publications, Inc., Mineola, NY 34. Fersht, A. (1977)in Enzyme Structure and Mechanism (Fersht, A., ed) Vol. 11,

pp. 69-71, W. H. Freeman and Co., New York 35. Gates, F. T.,111, and Linn, S. (1977)J. Biol. Chem. 262, 2802-2807 36. Katcher, H. L., and Wallace, S.S. (1983)Biochemistry 22,40714081 37. Gordon, L. K., and Haseltine, W. A. (1980)J. Biol. Chem. 266,12047-12050 38. Lu, A. L., and Chang, D.-Y. (1988)Genetics 118, 593-600 39. Lu, A. L., and Chang, D.-Y. (1988)Cell 64, 806-812 40. Au, K. G., Cabrera, M., Miller, J. H., and Modrich, P. (1988)P m . Natl. Acad. Sci. U. S. A. 86.9163-9166 41. Au,K G., Clark, S., Miller, J. H., and Modrich, P. (1989)Proc.Natl. Acad. Sci. U.S. A. 86,8877-8881 42. Michaels, M. L., Cruz, C., Grollman, A. P., and Miller, J. H. (1992)Proc. Natl. Acad. Sci. U. S. A. 89, 7022-7025 43. Kuo, C.-F., McRee, D. E., Cunningham, R. P., and Tainer, J. A. (1992)J. Mol. Biol. 227. 347-351 44. Kuo, C.-F., McRee, D. E., Fisher, C . L., OHandley, S. F., Cunningham, R. P., and Tainer, J. A. (1992)Science 268,434-440 45. %ai-Wu, J. J., Radicella, J. P., and Lu, A.-L. (1991)J. Bacteriol. 173, 19021910 ~

~

~~