ProteinâProtein Interactions Involving DNA Glycosylases - Chemical

OCTOBER 2003 VOLUME 16, NUMBER 10 © Copyright 2003 by the American Chemical Society

Invited Review Protein-Protein Interactions Involving DNA Glycosylases Bo Hang* and B. Singer* Life Sciences Division, Lawrence Berkeley National Laboratory, University of California, Berkeley, California 94720 Received April 22, 2003

Contents 1. Base Excision Repair: Pathways and Functions 2. Glycosylase-AP Endonuclease Interactions: Coupling of the First and Second Step in BER 2.1. Enhancement of Glycosylase Activities by AP Endonucleases 2.2. Protein-Protein Interaction Studies 2.3. Implications of the Glycosylase-AP Endonuclease Interaction 3. Interactions of DNA Glycosylases with Proteins from Other Repair Pathways: Relation to BER Initiation 3.1. Interaction between Glycosylases and NER Proteins 3.1.1. Functional Overlap between BER and NER Pathways 3.1.2. Protein-Protein Interactions between Glycosylases and NER Proteins 3.1.3. Transcription-Coupled BER of Oxidative Base Damage 3.1.4. Role of Accessory Proteins in BER and NER Interactions 3.2. Role of Glycosylases in Coordination of BER and MMR 3.2.1. Overlapping Specificities of Glycosylases and MMR

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3.2.2. Physical Interactions between Glycosylases, Accessory Proteins, and MMR Components 4. Perspective 5. References

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1. Base Excision Repair: Pathways and Functions BER1 is an important and often primary mechanism for removing DNA base modifications including oxidized, alkylated, exocyclic, and deaminated bases. These lesions can be formed by environmental agents or through internal metabolic processes (1-6). BER is also responsible for correcting certain base-base mismatches that originate either from miscoding during DNA replication/ recombination or from spontaneous processes. Another crucial role of BER is to repair cytotoxic and mutagenic AP sites resulting from spontaneous depurination and depyrimidination or as a reaction intermediate of BER. The spontaneous depurination, as measured by Lindahl and Nyberg (7) under in vitro conditions and extrapolated to a cellular genome, could produce AP sites at an estimated rate of ∼10 000 per day/per cell. The occurrence of such endogenous AP sites in mammalian genomic DNA represents one of the most abundant types * To whom correspondence should be addressed. (B.H.) Tel: 510495-2537. Fax: 510-486-6488. E-mail: [email protected]. (B.S.) Tel: 510-642-0637. Fax: 510-486-6488.

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Figure 1. BER subpathways in mammalian cells. GO, 7,8-dihydro-8-oxoG; G, 1,N2-ethenoG.

of DNA damage (8). This pathway clearly plays a role in counteracting the increased spontaneous mutation in prokaryotes and is likely to be a relevant factor in human cancer susceptibility (9-11). Thus, BER is a “housekeeping” system essential for the maintenance of genetic integrity. Mice that lack key components of BER, e.g., the major AP endonuclease, polymerase β (Pol β), or 1Abbreviations: BER, base excision repair; NER, nucleotide excision repair; MMR, mismatch repair; TCR, transcription-coupled repair; AP, apurinic/apyrimidinic; Tg, thymine glycol; 8-oxoG (GO), 7,8-dihydro8-oxoguanine; 5-mC, 5-methylcytosine; 3-mA, 3-methyladenine; DHU, 5,6-dihydrouracil; C, 3,N4-ethenocytosine; A, 1,N6-ethenoadenine; APNG, alkylpurine-DNA glycosylase; MPG, methylpurine-DNA glycosylase; TDG, thymine-DNA glycosylase; Mug, mismatch uracil-DNA glycosylase; UDG (UNG), uracil-DNA glycosylase; SMUG1, single strand selective monofunctional uracil-DNA glycosylase; MBD4, methyl-CpG-binding domain 4; MutY, mismatch adenine glycosylase; MYH, MutY homologue; OGG1, 8-oxo-G glycosylase 1; Endo III, endonuclease III; NTH1, Endo III homologue 1; Nei, endonuclease VIII; NEIL, Nei like homologue; APE1, AP endonuclease-1; Endo IV, endonuclease IV; Exo III, exonuclease III; Pol, polymerase; FEN1, flap endonuclease-1; XPG, xeroderma pigmentosum group G protein; CSB, Cockayne syndrome group B protein; PCNA, proliferating cell nuclear antigen; RPA, replication protein A; RF-C, replication factor-C; XRCC1, X-ray cross-complementation protein 1; YB-1, Y box-binding protein 1; EMSA, electrophoretic mobility shift assay.

XRCC1, do not survive embryogenesis (10, 12), which indicates the importance of BER for viability. Recent in vitro studies have revealed at least two subpathways in BER that differ in their repair patch sizes but share some common proteins. The details of such pathways have been reviewed in many recent articles (e.g., 2, 12-21). A BER pathway is initiated with a damage specific DNA glycosylase that recognizes and excises a modified or incorrect base (Figure 1). Two types of DNA glycosylases have been identified as follows: (i) monofunctional DNA glycosylases, which catalyze hydrolytic cleavage of the N-C1′ glycosylic bond between the target base and the sugar, producing an AP site and (ii) bifunctional DNA glycosylases, which, after glycosylase action, further cleave the DNA backbone on the 3′side of the AP site with the associated AP lyase activity. The Escherichia coli glycosylases are among the first glycosylases to be discovered. The genetics, biology, and biochemistry of these enzymes have been well-studied (2, 13, 16). In the past decade, it has been possible to clone many human DNA glycosylases by taking advantage of functional complementation with their E. coli or yeast

Invited Review

homologues or sequence homology searches through the human genome database. The human monofunctional glycosylases (in nucleus) include two UDGs (UNG2 and SMUG1), APNG (also called MPG or AAG), TDG, and the mismatch specific glycosylase MBD4. The well-known glycosylase/lyases identified in humans are NTH1 and OGG1. More recently, two novel human glycosylase/ lyases, which are homologous to E. coli Fpg/Nei, have been identified and characterized (22-25). These are termed NEIL1 (Nei like) (22-24) and NEIL2 (22, 25). NEIL3 could be the third candidate glycosylase (26). The human MYH, hMYH1, has a controversial or weak AP lyase activity (27-30). The AP site produced by a glycosylase is recognized and processed by a 5′-AP endonuclease, which cleaves the phosphodiester bond 5′ to the AP site, yielding a 3′-OH and 5′-deoxyribose phosphate (dRP) residue (2, 31-33). The AP endonuclease also processes the glycosylase/lyase product, a 3′-cleaved AP site, by excising the 3′-R,β-unsaturated aldehyde, thus providing a suitable primer for DNA polymerase. In the primary or socalled “short patch” BER (Figure 1), the single nucleotide gap is filled through template-directed synthesis by Pol β, which also uses its dRPase activity to remove the remaining 5′-dRP moiety at the AP site. Subsequently, the nick is sealed by DNA ligase I or by the ligase III/XRCC1 complex. In the long patch BER, which requires PCNA, Pol δ or Pol catalyzes the repair patch synthesis (2-8 nucleotides long) for displacing the AP site-containing strand. The resulting flaplike structure is removed by a structure-specific endonuclease, FEN1. The nick is probably sealed by DNA ligase I (Figure 1) (2, 12-21). It has been proposed that the type of DNA damage present in DNA and the type of responding glycosylases involved in repair, which generate different types of termini at the AP site, are important factors in the selection of a BER subpathway (34, 35). Most glycosylases appear to initiate the short patch BER pathway. These include UNG2 (36-39), SMUG1 (39), NTH1 (40, 41), and OGG1 (34, 42, 43). hMYH may be involved in long patch BER (44), and APNG appears to be implicated in both short and long patch pathways (34). It should be noted that the evidence for the distinct subpathways in mammalian cells is mainly based on biochemical results from reconstituted repair systems and cell-free repair assays (e.g., 45-49). BER of the oxidized bases, Tg and 8-oxoG, could be transcription-coupled (50-53); that is, the removal of these lesions from transcribed strands is more efficient than that from nontranscribed strands. This process appears to require specific glycosylase/lyases, XPG, CSB, and several other cellular proteins (50, 51, 53-55) (also see section 3.1.3). However, the detailed molecular mechanism for this pathway remains to be elucidated. One implication of this mechanism is that a defective TCR of endogenously formed oxidative DNA damage may contribute to the pathogenesis of Cockayne syndrome (CS) (50, 51, 53). Recently, Pol ι, one of the newly discovered UmuC/ DinB superfamily translesional DNA polymerases (56-58), was shown to have a dRPase activity and to be able to replace Pol β as an efficient short gap-filling Pol in vitro (59). Given the unique enzymological properties of Pol ι, such Pol ι-involved BER, if it exists in vivo, is likely to be used in certain special circumstances (59).

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One circumstance is that Pol ι inserts G opposite a template T generated by the deamination of 5-mC, thus preventing the base mutation (59, 60). Unlike NER, BER reactions performed in vitro generally do not require protein complex formation or cofactors, although complexes composed of two proteins, e.g., XRCC1-ligase III (61), or of multiple proteins (62) have been reported. However, in order for BER enzymes to perform sequential and concerted reactions, protein-protein interactions, direct or indirect, are required at each major step (18, 63). Many of these reactions are important for the modulation of the catalytic activities of the enzymes involved in BER. BER is also coordinated with other major repair pathways such as NER and MMR. In addition, BER couples with transcription and may also associate with DNA replication and cell cycle regulation. It is believed that proteinprotein interactions play a fundamental role in all of these connections. The purpose of this overview is to examine what is known about the protein-protein interactions involving the DNA glycosylases, and how such interactions modulate and relate to the functional linkage between a glycosylase and proteins in the BER pathway or from other repair pathways. Because of the rapid explosion of information in these related fields during past decade, we regret that it is not possible in this overview to cite all prior relevant publications.

2. Glycosylase-AP Endonuclease Interactions: Coupling of the First and Second Step in BER Glycosylases act on a DNA lesion via DNA-protein interactions, which is the only damage-specific step in BER (15, 18, 64-66). Crystal structures of many of the DNA glycosylases suggest that they employ a similar base-flipping mechanism prior to their catalytic activity (18, 64-69). It has been proposed that DNA glycosylases scan the DNA by slightly “pinching” the phosphodiester backbone, which would cause a distortion or kinking of the DNA at the base damage site. With additional pushing by the enzyme, the target nucleotide is flipped out of the DNA double helix and enters a specific active site pocket in the enzyme. Certain glycosylases such as human UNG and E. coli Mug are known to flip an AP site out (70, 71). AP endonucleases can similarly cause DNA kinking and facilitate AP site sugar flipping (72, 73). These structural or mechanistic features of glycosylase-DNA and AP endonuclease-DNA interactions are thought to be important for certain glycosylase-AP endonuclease interactions (63, 70, 73).

2.1. Enhancement of Glycosylase Activities by AP Endonucleases AP endonuclease is a pivotal enzyme in BER and is at the “intersection” of all of the glycosylases (32, 33). In the past few years, many researchers have investigated both the functional and the physical interactions between DNA glycosylases and AP endonucleases. Before or during this period, direct interactions were reported between other BER component proteins, including AP endonuclease, Pol β, PCNA, RPA, FEN1, XRCC1, and DNA ligases (e.g., 37, 61, 62, 74-76). In 1998, Parikh et al. (70) described an enhancement of hUNG activity by


Figure 2. Hand-off of damage intermediates between UDG and damage-general BER. Reprinted with permission from ref 70. Copyright 1998 Oxford University Press.

the human APE1 (also called HAP1, APEX, or Ref-1). From the combined crystallographic and biochemical data (70, 77), it was suggested that the mechanism for such enhancement appears to be the forced release of hUNG from its product AP site by APE1. hUNG binds to the U:G-DNA through the minor groove but not extensively (70). The glycosylase remains bound to the AP site or possibly rebinds, thus inhibiting the enzymatic turnover. In contrast, APE1 has a more extensive AP-DNA minor groove binding and causes more DNA bending than does hUNG (72, 73). On the basis of these findings, the authors proposed a primarily structure-based “hand-off” model (Figure 2) and hypothesized that a similar mechanism may be used for other glycosylases: (i) UNG detects and cleaves uracil from DNA and remains bound to the product AP site and (ii) APE1 processively scans the DNA and pries UNG off the AP site as a result of its extensive binding to the minor groove and greater distortion of the bound DNA. This mechanism seems to efficiently couple the first damage specific step with the second damage general step in BER (70). Many later studies have confirmed that this “coupling” is a more general phenomenon among other glycosylases (Table 1). Similar to UNG, these glycosylases usually bind tightly to their final reaction products, an AP site, or a 3′-cleaved AP site, and the rate-limiting step is the turnover of the bound enzyme. From the literature, it seems that a specific mechanism(s) involving AP endonuclease is required to facilitate the release of such a

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bound glycosylase, although the mechanism could vary for different glycosylases. In contrast to the kinetically efficient UNG (82), most glycosylases function with much lower efficiencies. Two extreme examples are hTDG and its bacterial homologue, Mug, both of which are virtually single turnover enzymes because of their extremely tight binding to the AP site (83-85). The stimulation of hTDG (84-86) or Mug (87, 88) by AP endonuclease appears to be the result of their competition for the same AP site, since (i) catalytically active Endo IV is required for Mug stimulation (87), implying that the dissociation of Mug is dependent on AP site cleavage; (ii) the stimulatory effect is dependent on the concentrations of AP endonuclease, and significant glycosylase turnover was observed only with a high molar excess of AP endonuclease (84); and (iii) no direct hTDG-APE1 interaction is identified using various methods, including the two-hybrid system (85). Interestingly, the covalent modification of hTDG by the ubiquitinlike proteins potentiates the APE1 stimulation on hTDG (86). Such modification itself greatly reduces the TDG affinity for the AP site, thus facilitating the enzyme turnover (86). The mammalian bifunctional glycosylases OGG1 remove 8-oxoG with low efficiency (89-91). The AP site is the major product of OGG1 and the rate-limiting step is its lyase activity (89, 91). Human APE1 can significantly stimulate the hOGG1 activity in vitro as shown in three independent studies (91-93), and the mechanism for such stimulation could be attributed to more than one factor. hOGG1 has a much higher affinity for the product AP site (AP:C) than for the substrate 8-oxoG:C, suggesting that it remains tightly bound to the AP site (91). However, APE1 cleaves the AP site at a rate much faster than that of the AP lyase activity of hOGG1 (91), which increases the hOGG1 turnover by reducing the AP sites (hOGG1 does not bind to the 5′-incised AP site (92)). In addition, the binding kinetics combined with catalytic mutant studies (91, 92) suggest that APE1 also competes with hOGG1 for AP site binding, thus facilitating hOGG1 turnover and eliminating the AP lyase step. In the absence of APE1, OGG1 may remain bound to the AP site and/or its final product 3′-cleaved AP site (91, 92). There are conflicting data on whether APE1 stimulates the AP lyase activity of hOGG1 (92, 93). In one study, a molar excess of APE1 enhanced the AP lyase activity of GST-OGG1 protein near 2-fold (93), which could increase the OGG1 turnover. Most recently, it was also reported that the activity of another human DNA glycosylase/ lyase, hNTH1, which belongs to the same super family with OGG1 (Endo III superfamily), could be stimulated by human APE1 (94). Interestingly, such stimulation by APE1 is dependent on the nature of the base opposite Tg in DNA (94). APE1 stimulates hNTH1 activity against Tg:A by abrogation of the AP lyase activity, in a way similar to the stimulation of hOGG1 by APE1 as described before (91, 92). However, when Tg pairs with G, APE1 has no effect on the AP lyase activity of hNTH1 or on its glycosylase activity against Tg:G under turnover conditions (94). Kinetically, the NTH1 glycosylase activity toward Tg:A is much higher than its AP lyase activity, whereas the enzyme shows concomitant glycosylase and AP lyase activities when Tg:G is the substrate (94-96). In addition, the AP lyase activity of hNTH1 is much higher toward AP:G than toward AP:A (97). It appears that the differential activity of hNTH1 and differential

Invited Review

Chem. Res. Toxicol., Vol. 16, No. 10, 2003 1185 Table 1. Enhancement of Glycosylase Activities by AP Endonucleases or Other Proteins

glycosylase studied

substrate used

hUDGs T. cruzi UDG

U (in DNA)

hSMUG1

U (in ssDNA and dsDNA) U:G, T:G,C:G

hTDG E. coli Mug hOGG1

U:G, C:G, 8-hm-C:G 8-oxoG:C

hNTH1

Tg:A DHU

M. thermoautotrophicum Endo III mouse Myh

Tg:A

E. coli MutY

8-oxoG:A G:A G:A

AP endonuclease that stimulates glycosylase activity

protein that stimulates glycosylase activity

APE1 (70, 78) L. major AP endo Exo III (79) APE1 (80, 81)

protein that shows no effect on glycosylase activity XPG (40) APE2, FEN1 (81)

APE1 (84-86)

SUMO-1, SUMO-3 (86)

Pol β, XRCC1, ligase III (84); E. coli Nfo, Nth, fpg (85)

Endo IV (87, 88) APE1 and catalytic mutants, Endo IV (91-93) APE1 (94)

XPG (40) XPG (40, 99) YB-1 (95)

M. thermoautotrophicum Endo IV (100) APE1 and catalytic mutant (102) Exo III, Endo IV (101)

stimulatory effect by APE1 in processing of Tg:A vs. Tg:G are a function of the affinity of hNTH1 for different Tg and AP site base pairs (95). hNTH1 can also be stimulated in vitro, regardless of base pairing, by YB-1 (95), a transcription factor that also interacts with PCNA (98). In addition, XPG protein can strongly stimulate hNTH1 activity in in vitro assays (40, 99) (see section 3.1.3). A similar AP endonuclease-mediated stimulation has been observed with E. coli MutY (101) and its murine homologue, mMyh (102), two glycosylases with no significant AP lyase activity (27-30). They both remove A when mispaired with 8-oxo-G or G, and the rate-limiting step is the release of the product AP site. The latter would cause a problem since these two glycosylases lack an efficient AP lyase activity, which would result in the persistence of AP sites in the absence of a 5′-AP endonuclease. APE1 stimulates both 8-oxoG:A and G:A activity of mMyh (102), while the E. coli AP endonucleases, Exo III and Endo IV, only enhance the G:A activity of MutY but not its 8-oxoG:A activity (101).

2.2. Protein-Protein Interaction Studies Given the ability of AP endonucleases to stimulate many glycosylases, a basic question is whether these two types of enzymes interact directly. Most of the studies thus far have failed to show a physical glycosylase-AP endonuclease interaction using various techniques including EMSA (70, 84-87, 91-93), coimmunoprecipitation (78), protein mutants (91, 92, 102), and yeast twohybrid system (85). The only exception appears to be hMYH, for which a direct interaction with human APE1 has been demonstrated by immunoprecipitation, affinity binding, and Western blotting (44). The APE1 binding site on hMYH was found in the region of residues 295318 using constructs containing different portions of hMYH fused to GST (44). Although a functional interaction has not yet been reported between these two human proteins, human APE1 does stimulate mMyh (102), which shares 78% homology with hMYH. For E. coli MutY, a super-shifted band was observed in EMSA of the combined reaction of MutY and Exo III with G:A or 8-oxoG:A oligomer (101). In addition, a C-terminal trun-

PCNA, RPA, RFC, FEN1, XPA, XPC, hRad51, Pol β, ligase I, PK, p53 (40)

APE1, UDG, Pol I (101)

cated form of MutY is no longer stimulated by either Exo III or Endo IV (101). These data support a direct interaction between E. coli MutY and AP endonucleases. Taken together, it appears that a glycosylase-AP endonuclease interaction could occur indirectly such as competition for the same AP site (78, 84, 85, 91, 92) or could be direct as has been suggested for hMYH and MutY (44, 101).

2.3. Implications of the Glycosylase-AP Endonuclease Interaction Although the physiological significance of the glycosylase-AP endonuclease interactions remain to be elucidated, there may be at least two potential implications of such interactions: first, it facilitates the efficiency of the glycosylase by reducing the product inhibition. In vivo, this may be particularly useful for those “poor substrates” as defined from in vitro experiments and for those glycosylases that are not inducible. The question is (i) would such enhancement be significant at the physiological concentrations of the AP endonuclease(s) or (ii) while multiple glycosylases are in action, would the cell have sufficient AP endonuclease to function? This appears not to be a problem for certain types of human cells such as HeLa cells, which have abundant APE1 with estimated concentrations at least in the micromolar range (103, 104). On the other hand, a recent study using human fibroblast cell-free extracts showed limited differences in amounts between APE1 and hOGG1 or hUNG (105). There is evidence that mammalian AP endonuclease is inducible under oxidative stress (93, 106, 107). In such a case, more APE1 could presumably be available for the stimulation of those glycosylases repairing oxidative damage. The second function of this interaction, which may be more important, seems to be to protect the AP site, which can be cytotoxic by blocking polymerases or mutagenic due to its noncoding nature. The AP site itself is also labile with a tendency to form DNA strand breaks (108). In many cases, DNA glycosylases actually convert less harmful base alterations into a more lethal and mutagenic lesion, i.e., the AP site. An imbalanced BER could


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also occur when DNA glycosylase(s) is overexpressed, leading to the accumulation of AP sites or strand breaks (109-112), which can be largely suppressed by the cooverexpression of an AP endonuclease or enhanced by the underexpression of the AP endonuclease (113). It is proposed that the tightly bound glycosylase at the AP site stabilizes and protects the AP site and that the enzyme itself may also serve as a signal for recruiting to the site an AP endonuclease or other proteins in a timely manner. Such an interaction mode was recently proposed to explain how, in general, BER enzymes conduct sequential reactions (“passing the baton”) (18, 63).

3. Interactions of DNA Glycosylases with Proteins from Other Repair Pathways: Relation to BER Initiation In general, major repair pathways including BER, NER, and MMR have their own substrate specificities and their own sets of enzymes/proteins. However, an increasing amount of evidence, obtained in vitro and in vivo, has demonstrated substantial functional overlapping among these pathways. Although it is still unclear to what degree these pathways overlap in vivo and what the exact regulatory mechanisms are, many proteinprotein interactions have been identified in vitro and some have been proven to be essential for a functional linkage. These interactions could take place either (i) directly between two major component proteins of different pathways, e.g., human glycosylase APNG (BER) and human RAD23 homologues (NER) (114), or (ii) through cellular accessory proteins such as PCNA and RPA that are architecturally and functionally important to many repair proteins (115-117). In the following sections, we describe the interactions of NER and MMR with BER, with a focus on the BER glycosylases. It should be noted that overlapping specificities and interplay have also been identified between NER and MMR pathways. Thus far, the major proteins from each step of BER (Figure 1) are known to be able to interact with proteins inside and/or outside the pathway. For a DNA glycosylase, such interactions may be particularly or uniquely important, since (i) it catalyzes the first rate-limiting step in BER, which means that any enhancement of its activity is likely to increase the efficacy of the whole pathway, (ii) its activity could be an important determinant of the equilibrium between BER and the other overlapping repair pathway, and (iii) coupling of BER to transcription or replication or any attempt to colocalize a BER pathway logically has to start with its initial enzyme; i.e., a DNA glycosylase. Moreover, such coupling action must fit with the specificity of BER, which is only conferred by DNA glycosylases.

3.1. Interaction between Glycosylases and NER Proteins The NER pathway removes the majority of bulky distorting lesions (2, 17, 118-121). The basic steps of human global genome NER include (i) damage recognition by XPC-hHR23B; (ii) multiprotein complex formation at the damage, which includes XPC-hHR23B, TFIIH (contains two helicases, XPB and XPD), XPA, and RPA; (iii) dual incision on two sides of the damage by structure specific endonucleases XPG (3′) and XPF/ERCC1 (5′); (iv) resynthesis to fill the resulting gap by the action of Pol

Figure 3. Summary of identified protein-protein interactions between BER and NER pathways. The dotted line indicates no direct evidence of a protein-protein interaction.

δ or , which requires PCNA; and (v) ligation performed by DNA ligase I (see Figure 3 right).

3.1.1. Functional Overlap between BER and NER Pathways Various in vitro studies have shown that NER can also act on certain nonbulky base lesions, most of which are substrates for the BER pathway (e.g., 122-127). One particular area that has been found to have such overlap is in the processing of oxidative DNA damage. Two of the major lesions in this class, Tg and 8-oxoG, have been shown to be recognized and removed from DNA by either E. coli (A)BC excinuclease (122-124) or the human NER system in vitro (127). However, whether human NER is significantly involved in the cellular removal of such lesions is questionable, considering that the in vitro repair rate of NER toward Tg or 8-oxoG is fairly slow (42, 127). In addition, the AP site, an intermediate from BER, can also be the substrate for both E. coli (122, 125) and human NER systems (126). In vivo studies, carried out only in yeast, have shown the evidence of functional interplay between BER and NER in the processing of oxidative, alkylated, and spontaneous DNA damage (128-132). In one study in Saccharomyces cerevisiae, the additional disruption of NER in the triple mutant of BER proteins Ntg1p, Ntg2p (NTH homologues), and Apn1p (the predominant AP endonuclease) caused a synergistic increase in mutation rates relative to each single mutant (128). Two other studies using yeast BER and NER double mutants (129, 130) also showed that both pathways contribute to the alkylation resistance and that a synergistic interaction is present between Mag1-initiated BER and the yeast NER genes (129, 130). It is generally thought that different repair pathways may play a backup role in repair of certain lesions, which may partially explain the lack of or mild phenotypic

Invited Review

change(s) in mutant mice without an individual DNA glycosylase (96, 133-139). Another common source for such backup activity could be from the DNA glycosylase(s) that has overlapping substrate specificity (135, 139141). For example, in the liver extract of NTH1 knockout mice, there are at least three separate Tg-DNA glycosylase backup activities (139, 140), one of which is the newly discovered glycosylase NEIL1 (140, 141). In contrast, in APNG knockout mice, it appears that mouse APNG is the sole glycosylase responsible for the removal of 3-mA, A, and hypoxanthine (134, 142).

3.1.2. Protein-Protein Interactions between Glycosylases and NER Proteins Many biochemical studies have shown that BER and NER pathways are closely related via multiple, direct protein-protein interactions (Figure 3). Several DNA glycosylases are found to interact with protein(s) involved in NER, thus providing a potential way for modulation of the BER pathway from its first step. Recently, Miao et al. (114) found that human glycosylase hAPNG physically interacts with the human Rad23 homologues, hHR23A and hHR23B. The latter protein forms a complex with XPC (XPC-HR23B), which can specifically bind to certain DNA lesions and initiate a NER reaction in vitro (120, 143-145). hHR23B binds to the hAPNG and stimulates its activity by increasing the affinity of hAPNG for substrates (114). Also recently, using a yeast two-hybrid system, Shimizu et al. (146) described a direct interaction between XPC and the human glycosylase hTDG. A significant in vitro stimulation of hTDG activity can be achieved in the presence of XPC-HR23B (146). These results suggest that hHR23 proteins or XPCHR23B may play a role in modulating the initial steps of both pathways (see Figure 3). Whether these NER proteins only specifically interact with and facilitate certain type(s) of DNA glycosylase remains to be determined, as does whether these interactions have any physiological roles.

3.1.3. Transcription-Coupled BER of Oxidative Base Damage Another functional interaction discovered in vitro recently is between hNTH1 and XPG (40, 99). The latter is a structure-specific endonuclease, which incises damaged DNA 3′ to the lesion and is also required nonenzymatically for subsequent 5′-incision by XPF/ERCC1 in NER (147-150). It is a homologue of the BER enzyme, FEN1, also a structure-specific endonuclease cleaving the flaplike structure (151, 152). In 1997, Cooper et al. (50) discovered that cells from severely affected XPG/CS patients, but not XPG patients, have both reduced TCR and global genome repair (GGR) of the oxidized base Tg. These results point to a different function of XPG, since its nuclease activity is defective in both XPG and XPG/ CS cells (50). Inasmuch as Tg is primarily repaired by the BER pathway (41) and only those XPG/CS patients have severely truncated XPG proteins (153), it was speculated that XPG could facilitate glycosylase-mediated Tg removal through protein-protein interactions (50). Two later studies showed that, in vitro, XPG is able to enhance binding of hNTH1 to the Tg-containing substrate and to stimulate its glycosylase activity by 2-6-fold (40, 99). Such an effect can be achieved with the full-length


XPG nuclease catalytic site mutants (40, 99). However, the lack of overt phenotypic abnormalities in the NTH1 null mice (96, 139) does not support the notion that NTH1 may play a significant role in the TCR of Tg. NTH1 may mainly be involved in GGR of Tg, and its deficiency may not be enough to cause a marked phenotypic change (26). The search for a candidate glycosylase(s) responsible for the TCR of Tg should lead to a better understanding of this complex process. Similar to Tg, base excision removal of 8-oxoG from a mammalian genome could also be transcription-coupled (51-53). BER is the primary pathway for repair of 8-oxoG in mammalian cells (42). OGG1, which appears to be the major BER activity removing 8-oxoG, at least shown in extracts of ogg1-/- null mouse tissues (136), is actually not required for TCR of 8-oxoG but rather for its repair in nontranscribed sequences (51, 52). These results suggest that a glycosylase other than OGG1 is responsible for the TCR of 8-oxoG. One of such candidates could be the NEIL glycosylases, as have recently been postulated (26, 154). The TCR process requires the presence of several factors such as XPG, TFIIH (XPB and XPD), CSB, and BRCA1 and 2 (50, 51, 53, 55). In general, it is thought that TCR recruits BER enzymes to the transcription-blocking lesions such as Tg and 8-oxoG and displaces the stalled RNA polymerase II. So far, transcription-coupled BER is only found with oxidized bases that are usually excised by DNA glycosylase/lyases.

3.1.4. Role of Accessory Proteins in BER and NER Interactions BER and NER pathways could also cooperate through interactions mediated by accessory proteins such as PCNA and RPA. Some of these connections appear to involve glycosylases. PCNA has been well-known as a “cellular coordinator” or “molecular adaptor” since it interacts through specific binding with proteins from many cellular processes including BER, NER, MMR, DNA replication, and transcription (115-117). Recently, polymerases involved in translesion DNA synthesis were also added to this list (155-157). PCNA is a ring-shaped trimetric protein, and its basic function is serving as a “sliding clamp”. PCNA requires replication factor C (RFC), a “clamp loader”, which loads the PCNA onto DNA molecules (115-117). PCNA is required for long patch BER. It directly interacts and stimulates several key proteins including FEN1, Pol δ/, and ligase I (115-117, 158) (Figure 3). Recently, several DNA glycosylases have been shown to physically interact with PCNA. These are human UNG2 (159) and UDG from Archaea (160), human 5-mC-DNA glycosylase (161), and human and Schizosaccharomyces pombe MYH (44, 162). Their interactions are through a well-conserved consensus PCNA-binding motif, QXX(I/ L/M)XX(F/H)(F/Y), found in these glycosylases except for human 5-meC-DNA glycosylase. A recent in vivo study (162) showed that the yeast cell expressing a S. pombe MYH mutant, which retains normal glycosylase activity but cannot interact with S. pombe PCNA, is partially defective. These data support the functional importance of the PCNA-mediated interaction. PCNA is also required for the NER resynthesis (115-117). In addition, it directly interacts with XPG, although the specific function of such interaction is not fully understood (40, 163). In any case, one of the PCNA functions appears to recruit


these proteins to the site of DNA repair, providing a common ground for potential interactions between BER and NER proteins. Another possible interaction between BER and NER could be through RPA, a single-stranded DNA-binding protein required for DNA replication (164, 165). RPA is shown to physically interact with XPA (166-168), XPG (166, 169), and XPF/ERCC1 (169). The RPA mutant lacking the C terminus leads to the inhibition of NER activity in vitro (170). RPA is also shown to interact directly with two human glycosylases, UNG2 (159, 171, 172) and MYH (44), via a specific binding motif, in addition to its ability to stimulate final steps of long patch BER (173-175). On the basis of the findings that both UNG2 and MYH colocalize with PCNA and RPA at the replication foci (159, 176), it is postulated that these two glycosylases are involved in replication-associated repair (RAR). There is evidence that the levels of both glycosylases in nuclei are cell cycle-dependent with maximum levels in the S phase (176-178). Similar cell cycle dependence was also observed with PCNA (176). These data also support the role of UNG2 and MYH in RAR, i.e., to excise from the nascent strand a misincorporated uracil (U:A) or adenine (A:8-oxoG), respectively.

3.2. Role of Glycosylases in Coordination of BER and MMR The MMR pathway is mainly involved in postreplication repair by correcting single base-base mispairs from miscoding and short loops from insertion/deletion (2, 179-184). Defects in MMR have been associated with hereditary nonpolyposis colorectal carcinomas (179, 182, 184). The steps of E. coli MutHLS system have been wellcharacterized. Briefly, MutS protein recognizes and binds to a base mismatch. MutL then binds to MutS, and the MutS-MutL complex activates the endonuclease activity of MutH, which nicks the newly synthesized, unmethylated strand. UvrD helicase then unwinds the nicked DNA past the mismatch. The resulting single strand is degraded by a single-strand specific exonuclease, and the gap is filled through resynthesis. In eukaryotic cells, homologues of MutS and MutL are in heterodimers. There are two MutS homologues, MSH2/MSH6 and MSH2/MSH3, each with different mispair recognition specificity. Several MutL homologue dimers have been identified as follows: MLH1/PMS1 (yeast) or PMS2 (human), MLH1/PMS1 (human), and MLH1/MLH3. Exo 1 and FEN1 are two nucleases that may be involved in the excision step. The resynthesis requires Pol δ, PCNA, RFC, and RPA.

3.2.1. Overlapping Specificities of Glycosylases and MMR Biochemical studies have shown that E. coli MutS protein binds to several known glycosylase substrates such as a G:T mismatch (185, 186), 5-formyluracil (187), and A (185). The yeast MSH2-MSH6 complex can specifically and efficiently bind to a DNA containing the mismatches, 8-oxoG:A or 8-oxoG:C (188). The former is a substrate for glycosylases MutY/MYH, which excise the mispaired A opposite 8-oxoG (16, 19). The OGG1 glycosylases efficiently remove 8-oxoG from 8-oxoG:C (11, 19, 132). Similarly, the human MSH2-MSH6 complex, but not MSH2-MSH3, is also able to recognize 8-oxoG base

Hang and Singer

pairs in DNA, particularly those mismatched ones (189). In addition to these glycosylase substrates, MMR recognition proteins also bind to a number of other DNA lesions such as 1,N2-propanoG (186), malondialdehydedG adduct (186), alkylated bases (190, 191), and cisplatin-DNA adducts (192-194). One caveat to the binding specificity of MMR discussed above is that such binding does not necessarily indicate a role of MMR in the cellular repair of these lesions. In some cases, the recognition/ binding by MMR proteins may interfere with other repair mechanisms (21). Genetic evidence suggests that MMR plays a role in counteracting the mutagenicity of the oxidative base damage (132, 188, 195-197). One study using S. cerevisiae has shown that the double mutants of msh2 (or msh6 or mlh1) and ogg1 caused a synergistic increase in mutation rates (188). Analysis of the types of base pair mutation in the MMR deficient yeast stains (188, 195) strongly suggests that MMR is the major mechanism in eliminating adenine incorporated opposite 8-oxoG. This is consistent with the finding that MSH2-MSH6 has a high affinity for 8-oxoG:A mispair (188). Therefore, MMR serves as a functional homologue of MutY since MutY is absent in yeast. However, it is still unclear regarding the role of MSH2-MSH6-dependent MMR in repair of such lesions in other species that possess a MYH. In a study (196) using mouse embryonic stem cells with a deficient Msh2, a significantly greater amount of 8-oxoG was accumulated in genomic DNA of the irradiated stem cells as compared to wild-type cells, indicating a reduced level of DNA repair.

3.2.2. Physical Interactions between Glycosylases, Accessory Proteins, and MMR Components Several DNA glycosylases have been demonstrated to interact directly with MMR proteins. hMYH was found to interact with the MSH6 protein in the MSH2-MSH6 complex and its binding and glycosylase activity toward 8-oxoG:A could be stimulated by MSH2-MSH6 (198). Reversely, hMYH did not have an effect on the binding of MSH2-MSH6 to DNA containing an 8-oxoG:A (198). This suggests that the hMYH-mediated BER may target 8-oxoG:A more efficiently in the presence of MMR. Moreover, the interaction of hMYH with MSH2-MSH6 may help to target hMYH to the newly synthesized DNA strand to remove adenine incorporated opposite 8-oxoG (198). Additionally, both hMYH and hMSH6 physically interact with PCNA and colocalize with PCNA to replication foci (44, 176, 199, 200), further suggesting that these two pathways that have similar functions in postreplication repair may cooperate with each other in reducing replication errors originated from 8-oxoG. In general, because both PCNA and RPA are also implicated in MMR and interact with many proteins in the pathway (e.g., 199-207), these two accessory proteins could potentially coordinate BER and MMR through multiple connections. Using yeast two-hybrid screening and in vitro GST pull-down experiments, a physical interaction was observed between yeast glycosylase Ntg2p and Mlh1p (208), a key component in MMR (209-211). Genetic studies to address the biological relevance of this Ntg2-Mlh1p interaction showed that deletion of NTG2 did not affect MMR, but overexpression of Ntg2p led to a MMR mutator

Invited Review


Figure 4. Schematic presentation of various binding domains identified on human glycosylase hMYH.

phenotype that suggests a partial inhibition of MMR (208). Such inhibition could be the result of sequestration of Mlh1p by overproduced Ntg2p, thus preventing the interactions of Mlh1p with other protein partners in MMR (208). These data strongly suggest that the levels of expression of repair genes such as Ntg2 and Mlh1 may be tightly regulated to ensure the balance between two repair pathways (208). Human glycosylase MBD4 (also called MED1), which, like Ntg2p, belongs to the Endo III superfamily, is shown to directly interact with the human MLH1 protein (212). Transfection of a MBD4 mutant lacking the methyl-CpGbinding domain is associated with microsatellite instability (MSI) (212), a hallmark of a defective MMR (179). It is therefore proposed that MBD4 may be involved in MMR and in the maintenance of genomic stability (212). In supporting this, the MBD4 gene is found to be frequently mutated in human colorectal carcinomas with MSI (213).

4. Perspective DNA glycosylases have, at a minimum, the following functions: (i) recognizing specific DNA lesions via DNA-protein interactions; (ii) catalyzing the hydrolysis of the glycosydic bond between the base and the sugar; (iii) cleaving the AP site at the 3′-side (AP lyase activity); (iv) protecting the labile AP site by remaining bound to the AP site and possibly by recruiting AP endonuclease or other enzymes to the site; and (v) participating in the coordination of BER with other repair pathways (e.g., NER, MMR) and cellular functions such as replication and transcription. In the past few years, much progress has been made in understanding the functions described in functions iv and v. In addition, certain DNA glycosylases are also known to interact with other proteins, which may have undetermined function (not necessarily repair-related) and importance. For example, TDG protein interacts with several transcription factors, suggesting a role of this glycosylase in the regulation of transcription (214-216). Until recently, DNA glycosylases were viewed as small monomeric proteins capable of carrying out in vitro reactions without a requirement for cofactors. At present, we envision these enzymes as those appear to be tightly coordinated and well-connected with proteins from the same as well as from other repair and cellular pathways. The physical evidence for this is clearly shown in the sequence of these glycosylases. One example is the wellstudied hMYH, a 59 kDa protein with a number of protein binding domains found in its structure. As shown in Figure 4, hMYH directly interacts with human APE1 utilizing the region 295-318 (44). This interaction is presumably important for hMYH both in enhancing its glycosylase activity and in timely processing of the cytotoxic/mutagenic AP site. hMYH also

contains a domain (residues 232-254) for interacting with the MMR initiating complex hMutSR (MSH2MSH6). In vitro, such interaction can lead to enhanced binding and glycosylase activity of hMYH toward 8-oxoG:A in DNA, suggesting that the efficiency of hMYH-initiated BER can be modulated by MMR (198). The hMYH sequence also contains PCNA and RPA binding domains at the termini (44). Interaction of hMYH with these accessory proteins provides a potential connection to NER, MMR, and replication. hMYH has different isoforms, which are produced by alternative splicing and localize to nuclei and mitochondria (176, 217, 218). These proteins may use the putative nuclear localization signal or mitochondrial targeting signal located on either terminal (not shown in Figure 4) to facilitate their subcellular localization (219). Such sequences have also been found for glycosylases hUNG (220), hOGG1 (219, 221), and hNTH1 (219). Glycosylases are lesion-specific or function-specific. In analyzing a glycosylase-protein interaction, a conclusion obtained from an individual glycosylase may be applicable to all or only to a group of glycosylases or may be just to the enzyme studied. One example is that among the several DNA glycosylases removing uracil from DNA, hUNG2 appears to be the only one so far that is found to specifically localize in the replication foci in the S phase (81, 135, 159). In any case, it is reasonable that glycosylases and other repair proteins could interact, associate, or coordinate with each other according to the specific cellular changes imposed by DNA damage, which inevitably should be the major determinant for such a complex process. For instance, hUNG2 and hMYH are two glycosylases so far found to interact physically and colocalize with proteins involved in the replication machinery during the S phase. The biological basis for this is that U and A (opposite G or 8-oxoG) are the misincorporated bases during replication, which need to be repaired immediately. Because MMR is also involved in postreplication repair of base-base mismatches, it is not surprising to see functional and physical interactions between hMYH and hMutSR (198) and the latter also interacts and colocalizes with PCNA to the replication foci (199, 200). The direct glycosylase-protein or protein-protein interactions certainly provide a basis for a functional linkage between proteins or pathways. Moreover, certain protein components could be shared by different pathways. However, although many protein-protein interactions have been identified and are useful in understanding mechanisms, functional interactions may not necessarily depend on direct interactions. Examples could include the stimulation of certain DNA glycosylases by an AP endonuclease as discussed in section 2. Another example is shown by the functional “cross-talk” between hOGG1 and CSB proteins in repair of 8-oxoG in a study


reported recently (222), in which a defective CSB causes a decreased rate of repair of 8-oxoG, which appears to be associated with down-regulated transcription of hOGG1 gene. There is no in vitro physical interaction between the two purified proteins (222), but transfection of the cells with wild-type CSB gene can complement the BER deficiency (223). So far, the regulatory mechanisms for many of these protein-protein or pathway-pathway interactions are unclear. In addition, such mechanisms could vary from prokaryotic to eukaryotic cells or even among eukaryotic organisms (224). Within the same species, tissue/organ specificity in enzyme activity/ expression and protein interactions is also present. Factors including gene regulation, chromatin structure, protein phosphorylation, etc. may all play a role in regulating these interactions. The glycosylase-protein and protein-protein interactions described in this overview have greatly enlarged our views on how different proteins or pathways can coordinate or associate to meet challenges. However, there is no doubt that in certain areas, studies on proteinprotein or pathway-pathway interactions have only begun or have not yet been initiated. One approach for addressing those questions of a protein-protein interaction in the cellular context is the use of gene knockouts or specific protein mutants, which may enable us to gain a greater understanding of the biochemical function and biological impact of those interactions between the target protein and the other DNA damage response proteins. Structure-functional studies of protein-protein, proteinintermediate-protein, and multiprotein complexes should provide more detailed biochemical and molecular details of these interactions. For example, the recently published three-dimensional structure of human RPA complex with a RPA binding domain of hUNG2 (172) provided a structural basis for understanding how RPA interacts and coordinates with proteins from different repair pathways such as UNG2, XPA, and RAD52. With further progress from these and other approaches, our understanding of the molecular mechanisms of such complex protein and pathway interactions, as well as their regulatory mechanisms, will expand. We anticipate that the DNA glycosylases, as BER initiating enzymes, will continue to be the focus of such future studies.

Acknowledgment. This work was supported by NIH Grants CA72079 (to B.H.) and CA47723 (to B.S.) and was administrated by the Lawrence Berkeley National Laboratory under Department of Energy contract DE-AC0376SF00098. We thank the reviewers for their valuable comments and useful suggestions.

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