Active site plasticity of endonuclease V from Salmonella typhimurium

Base deamination is a major type of DNA damage under nitrosative stress. Endonuclease V initiates repair of deaminated base damage by making a nucleolytic incision one nucleotide away from the 3' side of the lesion. Within the endonuclease V family, the substrate specificities are different from one enzyme to another. In this study, we investigated deamination lesion cleavage activities of endonuclease V from the macrophage-residing pathogen, Salmonella typhimurium. Salmonella endonuclease V exhibits limited turnover on cleavage of deoxyinosine- and xanthosine-containing DNA. Binding analysis indicates that this single-turnover property is caused by tight binding to nicked products. The nicking activity is similar between the double-stranded deoxyinosine- and deoxyxanthosine-containing DNA. Cleavage rates are not affected by bases opposite the deoxyinosine or deoxyxanthosine lesions. The enzyme is also active on single-stranded deoxyinosine- and deoxyxanthosine-containing DNA. Unlike endonuclease V from Thermotoga maritima, Salmonella endonucleae V can only turnover deoxyuridine-containing DNA to a limited extent when substrate is in excess. Binding analysis indicates that Salmonella endonuclease V achieves tight binding to deoxyuridine-containing DNA, a property that distinguishes it from Thermotoga endonuclease V. Cleavage analysis on mismatch-containing DNA also indicates that the active site of Salmonella endonuclease V can accommodate pyrimidine-containing mismatches, resulting in more comparable cleavage of pyrimidine- and purine-containing mismatches. This comprehensive DNA cleavage and binding analysis reveals the plastic nature in the active site of Salmonella endonuclease V, which allows the enzyme to enfold both purine and pyrimidine deaminated lesions or base pair mismatches.     

Mutational analysis of endonuclease V from Thermotoga maritima

Endonuclease V nicks damaged DNA at the second phosphodiester bond 3' to inosine, uracil, mismatched bases, or abasic (AP) sites. Alanine scanning mutagenesis was performed in nine conserved positions of Thermotoga maritima endonuclease V to identify amino acid residues involved in recognition or endonucleolytic cleavage of these diverse substrates. Alanine substitution at D43, E89, and D110 either abolishes or substantially reduces inosine cleavage activity. These three mutants gain binding affinity for binding to double-stranded or single-stranded inosine substrates in the absence of a metal ion, suggesting that these residues may be involved in coordinating catalytic metal ion(s). Y80A, H116A, and, to a lesser extent, R88A demonstrate reduced affinities for double-stranded or single-stranded inosine substrates or nicked products. The lack of tight binding to a nicked inosine product accounts for the increased rate of turnover of inosine substrate since the product release is less rate-limiting. Y80A, R88A, and H116A fail to cleave AP site substrates. Their activities toward uracil substrates are in the following order: H116A > R88A > Y80A. These residues may play a role in substrate recognition. K139A maintains wild-type binding affinity for binding to double-stranded and single-stranded inosine substrate, but fails to cleave AP site and uracil substrate efficiently, suggesting that K139 may play a role in facilitating non-inosine substrate cleavage.     

Dissecting endonuclease and exonuclease activities in endonuclease V from Thermotoga maritima

Endonuclease V is an enzyme that initiates a conserved DNA repair pathway by making an endonucleolytic incision at the 3′-side 1 nt from a deaminated base lesion. DNA cleavage analysis using mutants defective in DNA binding and Mn²⁺ as a metal cofactor reveals a novel 3′-exonuclease activity in endonuclease V [Feng,H., Dong,L., Klutz,A.M., Aghaebrahim,N. and Cao,W. (2005) Defining amino acid residues involved in DNA-protein interactions and revelation of 3′-exonuclease activity in endonuclease V. Biochemistry, 44, 11486–11495.]. This study defines the enzymatic nature of the endonuclease and exonuclease activity in endonuclease V from Thermotoga maritima. In addition to its well-known inosine-dependent endonuclease, Tma endonuclease V also exhibits inosine-dependent 3′-exonuclease activity. The dependence on an inosine site and the exonuclease nature of the 3′-exonuclease activity was demonstrated using 5′-labeled and internally-labeled inosine-containing DNA and a H214D mutant that is defective in non-specific nuclease activity. Detailed kinetic analysis using 3′-labeled DNA indicates that Tma endonuclease V also possesses non-specific 5′-exonuclease activity. The multiplicity of the endonuclease and exonuclease activity is discussed with respect to deaminated base repair.     

Multiple cleavage activities of endonuclease V from Thermotoga maritima: recognition and strand nicking mechanism

Endonuclease V is a deoxyinosine 3'-endonuclease which initiates removal of inosine from damaged DNA. A thermostable endonuclease V from the hyperthermophilic bacterium Thermotoga maritima has been cloned and expressed in Escherichia coli. The DNA recognition and reaction mechanisms were probed with both double-stranded and single-stranded oligonucleotide substrates which contained inosine, abasic site (AP site), uracil, or mismatches. Gel mobility shift and kinetic analyses indicate that the enzyme remains bound to the cleaved inosine product. This slow product release may be required in vivo to ensure an orderly process of repairing deaminated DNA. When the enzyme is in excess, the primary nicked products experience a second nicking event on the complementary strand, leading to a double-stranded break. Cleavage at AP sites suggests that the enzyme may use a combination of base contacts and local distortion for recognition. The weak binding to uracil sites may preclude the enzyme from playing a significant role in repair of such sites, which may be occupied by uracil-specific DNA glycosylases. Analysis of cleavage patterns of all 12 natural mismatched base pairs suggests that purine bases are preferrentially cleaved, showing a general hierarchy of A = G > T > C. A model accounting for the recognition and strand nicking mechanism of endonuclease V is presented.     

Switching base preferences of mismatch cleavage in endonuclease V: an improved method for scanning point mutations

Endonuclease V (endo V) recognizes a broad range of aberrations in DNA such as deaminated bases or mismatches. It nicks DNA at the second phosphodiester bond 3′ to a deaminated base or a mismatch. Endonuclease V obtained from Thermotoga maritima preferentially cleaves purine mismatches in certain sequence context. Endonuclease V has been combined with a high-fidelity DNA ligase to develop an enzymatic method for mutation scanning. A biochemical screening of site-directed mutants identified mutants in motifs III and IV that altered the base preferences in mismatch cleavage. Most profoundly, a single alanine substitution at Y80 position switched the enzyme to essentially a C-specific mismatch endonuclease, which recognized and cleaved A/C, C/A, T/C, C/T and even the previously refractory C/C mismatches. Y80A can also detect the G13D mutation in K-ras oncogene, an A/C mismatch embedded in a G/C rich sequence context that was previously inaccessible using the wild-type endo V. This investigation offers insights on base recognition and active site organization. Protein engineering in endo V may translate into better tools in mutation recognition and cancer mutation scanning.     

Mutational Analysis of Bacterial NAD^＋-dependent DNA Ligase： Role of Motif IV in Ligation Catalysis

The bacterial DNA ligase as a multiple domain protein is involved in DNA replication, repair and recombination. Its catalysis of ligation can be divided into three steps. To delineate the roles of amino acid residues in motif IV in ligation catalysis, site-directed mutants were constructed in a bacterial NAD^＋-dependent DNA ligase from Thermus sp. TAK16D. It was shown that four conserved residues （D286, G287, V289 and K291） in motif IV had significant roles on the overall ligation. Under single turnover conditions, the observed apparent rates of D286E, G287A, V289I and K291R mutants were clearly reduced compared with that of WT ligase on both match and mismatch nicked substrates. The effects of D286E mutation on overall ligation may not only be ascribed to the third step. The G287A mutation has a major effect on the second step. The effects of V289I and K291R mutation on overall ligation are not on the third step, perhaps other aspects, such as conformation change of ligase protein in ligation catalysis, are involved. Moreover, the amino acid substitutions of above four residues were more sensitive on mismatch nicked substrate, indicating an enhanced ligation fidelity.     

Catalytic mechanism of endonuclease v: a catalytic and regulatory two-metal model

The enzyme endonuclease V initiates repair of deaminated DNA bases by making an endonucleolytic incision on the 3' side one nucleotide from a base lesion. In this study, we have used site-directed mutagenesis to characterize the role of the highly conserved residues D43, E89, D110, and H214 in Thermotoga maritima endonuclease V catalysis. DNA cleavage and Mn(2+)-rescue analysis suggest that amino acid substitutions at D43 impede the enzymatic activity severely while mutations at E89 and D110 may be tolerated. Mutations at H214 yield enzyme that maintains significant DNA cleavage activity. The H214D mutant exhibits little change in substrate specificity or DNA cleavage kinetics, suggesting the exchangeability between His and Asp at this site. DNA binding analysis implicates the involvement of the four residues in metal binding. Mn(2+)-mediated cleavage of inosine-containing DNA is stimulated by the addition of Ca(2+), a metal ion that does not support catalysis. The effects of Mn(2+) on Mg(2+)-mediated DNA cleavage show a complexed initial stimulatory and later inhibitory pattern. The data obtained from the dual metal ion analyses lead to the notion that two metal ions are involved in endonuclease V-mediated catalysis. A catalytic and regulatory two-metal model is proposed.     

Cleavage of deoxyoxanosine-containing oligodeoxyribonucleotides by bacterial endonuclease V

Oxanine (O) is a deamination product derived from guanine with the nitrogen at the N¹ position substituted by oxygen. Cytosine, thymine, adenine, guanine as well as oxanine itself can be incorporated by Klenow Fragment to pair with oxanine in a DNA template with similar efficiency, indicating that oxanine in DNA may cause various mutations. As a nucleotide, deoxyoxanosine may substitute for deoxyguanosine to complete a primer extension reaction. Endonuclease V, an enzyme known for its enzymatic activity on uridine-, inosine- and xanthosine-containing DNA, can cleave oxanosine-containing DNA at the second phosphodiester bond 3′ to the lesion. Mg²⁺ or Mn²⁺, and to a small extent Co²⁺ or Ni²⁺, support the oxanosine-containing DNA cleavage activity. All four oxanosine-containing base pairs (A/O, T/O, C/O and G/O) were cleaved with similar efficiency. The cleavage of double-stranded oxanosine-containing DNA was ~6-fold less efficient than that of double-stranded inosine-containing DNA. Single-stranded oxanosine-containing DNA was cleaved with a lower efficiency as compared with double-stranded oxanosine-containing DNA. A metal ion enhances the binding of endonuclease V to double-stranded and single-stranded oxanosine-containing DNA 6- and 4-fold, respectively. Hypothetic models of oxanine-containing base pairs and deaminated base recognition mechanism are presented.     

Human endonuclease V as a repair enzyme for DNA deamination

The human endonuclease V gene is located in chromosome 17q25.3 and encodes a 282 amino acid protein that shares about 30% sequence identity with bacterial endonuclease V. This study reports biochemical properties of human endonuclease V with respect to repair of deaminated base lesions. Using soluble proteins fused to thioredoxin at the N-terminus, we determined repair activities of human endonuclease V on deoxyinosine (I)-, deoxyxanthosine (X)-, deoxyoxanosine (O)- and deoxyuridine (U)-containing DNA. Human endonuclease V is most active with deoxyinosine-containing DNA but with minor activity on deoxyxanthosine-containing DNA. Endonuclease activities on deoxyuridine and deoxyoxanosine were not detected. The endonuclease activity on deoxyinosine-containing DNA follows the order of single-stranded I>G/I>T/I>A/I>C/I. The preference of the catalytic activity correlates with the binding affinity of these deoxyinosine-containing DNAs. Mg(2+) and to a much less extent, Mn(2+), Ni(2+), Co(2+) can support the endonuclease activity. Introduction of human endonuclease V into Escherichia coli cells deficient in nfi, mug and ung genes caused three-fold reduction in mutation frequency. This is the first report of deaminated base repair activity for human endonuclease V. The relationship between the endonuclease activity and deaminated deoxyadenosine (deoxyinosine) repair is discussed.     

Uracil-DNA glycosylases SMUG1 and UNG2 coordinate the initial steps of base excision repair by distinct mechanisms

DNA glycosylases UNG and SMUG1 excise uracil from DNA and belong to the same protein superfamily. Vertebrates contain both SMUG1 and UNG, but their distinct roles in base excision repair (BER) of deaminated cytosine (U:G) are still not fully defined. Here we have examined the ability of human SMUG1 and UNG2 (nuclear UNG) to initiate and coordinate repair of U:G mismatches. When expressed in Escherichia coli cells, human UNG2 initiates complete repair of deaminated cytosine, while SMUG1 inhibits cell proliferation. In vitro, we show that SMUG1 binds tightly to AP-sites and inhibits AP-site cleavage by AP-endonucleases. Furthermore, a specific motif important for the AP-site product binding has been identified. Mutations in this motif increase catalytic turnover due to reduced product binding. In contrast, the highly efficient UNG2 lacks product-binding capacity and stimulates AP-site cleavage by APE1, facilitating the two first steps in BER. In summary, this work reveals that SMUG1 and UNG2 coordinate the initial steps of BER by distinct mechanisms. UNG2 is apparently adapted to rapid and highly coordinated repair of uracil (U:G and U:A) in replicating DNA, while the less efficient SMUG1 may be more important in repair of deaminated cytosine (U:G) in non-replicating chromatin.     

Mutational Analysis of Highly Conserved Residues in the Phage PhiC31 Integrase Reveals Key Amino Acids Necessary for the DNA Recombination

Background

Amino acid sequence alignment of phage phiC31 integrase with the serine recombinases family revealed highly conserved regions outside the catalytic domain. Until now, no system mutational or biochemical studies have been carried out to assess the roles of these conserved residues in the recombinaton of phiC31 integrase.

Methodology/Principal Findings

To determine the functional roles of these conserved residues, a series of conserved residues were targeted by site-directed mutagenesis. Out of the 17 mutants, 11 mutants showed impaired or no recombination ability, as analyzed by recombination assay both in vivo and in vitro. Results of DNA binding activity assays showed that mutants (R18A, I141A, L143A,E153A, I432A and V571A) exhibited a great decrease in DNA binding affinity, and mutants (G182A/F183A, C374A, C376A/G377A, Y393A and V566A) had completely lost their ability to bind to the specific target DNA attB as compared with wild-type protein. Further analysis of mutants (R18A, I141A, L143A and E153A) synapse and cleavage showed that these mutants were blocked in recombination at the stage of strand cleavage.

Conclusions/Significance

This data reveals that some of the highly conserved residues both in the N-terminus and C-terminus region of phiC31 integrase, play vital roles in the substrate binding and cleavage. The cysteine-rich motif and the C-tail val-rich region of phiC31 integrase may represent the major DNA binding domains of phiC31 integrase.     

Repair of deaminated base damage by Schizosaccharomyces pombe thymine DNA glycosylase

Thymine DNA glycosylases (TDG) in eukaryotic organisms are known for their double-stranded glycosylase activity on guanine/uracil (G/U) base pairs. Schizosaccharomyces pombe (Spo) TDG is a member of the MUG/TDG family that belongs to a uracil DNA glycosylase superfamily. This work investigates the DNA repair activity of Spo TDG on all four deaminated bases: xanthine (X) and oxanine (O) from guanine, hypoxanthine (I) from adenine, and uracil from cytosine. Unexpectedly, Spo TDG exhibits glycosylase activity on all deaminated bases in both double-stranded and single-stranded DNA in the descending order of X > I > U  O. In comparison, human TDG only excises deaminated bases from G/U and, to a much lower extent, A/U and G/I base pairs. Amino acid substitutions in motifs 1 and 2 of Spo TDG show a significant impact on deaminated base repair activity. The overall mutational effects are characterized by a loss of glycosylase activity on oxanine in all five mutants. L157I in motif 1 and G288M in motif 2 retain xanthine DNA glycosylase (XDG) activity but reduce excision of hypoxanthine and uracil, in particular in C/I, single-stranded hypoxanthine (ss-I), A/U, and single-stranded uracil (ss-U). A proline substitution at I289 in motif 2 causes a significant reduction in XDG activity and a loss of activity on C/I, ss-I, A/U, C/U, G/U, and ss-U. S291G only retains reduced activity on T/I and G/I base pairs. S163A can still excise hypoxanthine and uracil in mismatched base pairs but loses XDG activity, making it the closest mutant, functionally, to human TDG. The relationship among amino acid substitutions, binding affinity and base recognition is discussed.     

Endonuclease V protects Escherichia coli against specific mutations caused by nitrous acid

Endonuclease V (deoxyinosine 3'-endonuclease) of Escherichia coli K-12 is a putative DNA repair enzyme that cleaves DNA's containing hypoxanthine, uracil, or mismatched bases. An endonuclease V (nfi) mutation was tested for specific mutator effects on a battery of trp and lac mutant alleles. No marked differences were seen in frequencies of spontaneous reversion. However, when nfi mutants were treated with nitrous acid at a level that was not noticeably mutagenic for nfi(+) strains, they displayed a high frequency of A:T-->G:C, and G:C-->A:T transition mutations. Nitrous acid can deaminate guanine in DNA to xanthine, cytosine to uracil, and adenine to hypoxanthine. The nitrous acid-induced A:T-->G:C transitions were consistent with a role for endonuclease V in the repair of deaminated adenine residues. A confirmatory finding was that the mutagenesis was depressed at a locus containing N(6)-methyladenine, which is known to be relatively resistant to nitrosative deamination. An alkA mutation did not significantly enhance the frequency of A:T-->G:C mutations in an nfi mutant, even though AlkA (3-methyladenine-DNA glycosylase II) has hypoxanthine-DNA glycosylase activity. The nfi mutants also displayed high frequencies of nitrous acid-induced G:C-->A:T transitions. These mutations could not be explained by cytosine deamination because an ung (uracil-DNA N-glycosylase) mutant was not similarly affected. However, these findings are consistent with a role for endonuclease V in the removal of deaminated guanine, i.e., xanthine, from DNA. The results suggest that endonuclease V helps to protect the cell against the mutagenic effects of nitrosative deamination.     

A conserved motif of vertebrate Y RNAs essential for chromosomal DNA replication

Noncoding Y RNAs are required for the reconstitution of chromosomal DNA replication in late G1 phase template nuclei in a human cell-free system. Y RNA genes are present in all vertebrates and in some isolated nonvertebrates, but the conservation of Y RNA function and key determinants for its function are unknown. Here, we identify a determinant of Y RNA function in DNA replication, which is conserved throughout vertebrate evolution. Vertebrate Y RNAs are able to reconstitute chromosomal DNA replication in the human cell-free DNA replication system, but nonvertebrate Y RNAs are not. A conserved nucleotide sequence motif in the double-stranded stem of vertebrate Y RNAs correlates with Y RNA function. A functional screen of human Y1 RNA mutants identified this conserved motif as an essential determinant for reconstituting DNA replication in vitro. Double-stranded RNA oligonucleotides comprising this RNA motif are sufficient to reconstitute DNA replication, but corresponding DNA or random sequence RNA oligonucleotides are not. In intact cells, wild-type hY1 or the conserved RNA duplex can rescue an inhibition of DNA replication after RNA interference against hY3 RNA. Therefore, we have identified a new RNA motif that is conserved in vertebrate Y RNA evolution, and essential and sufficient for Y RNA function in human chromosomal DNA replication.     

A residue in MutY important for catalysis identified by photocross-linking and mass spectrometry

MutY is an adenine glycosylase in the base excision repair (BER) superfamily that is involved in the repair of 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG):A and G:A mispairs in DNA. MutY contains a [4Fe-4S]2+ cluster that is part of a novel DNA binding motif, referred to as the iron-sulfur cluster loop (FCL) motif. This motif is found in a subset of members of the BER glycosylase superfamily, defining the endonuclease III-like subfamily. Site-specific cross-linking was successfully employed to investigate the DNA-protein interface of MutY. The photoreactive nucleotide 4-thiothymidine (4ST) incorporated adjacent to the OG:A mismatch formed a specific cross-link between the substrate DNA and MutY. The amino acid participating in the cross-linking reaction was characterized by positive ion electrospray ionization (ESI) tandem mass spectrometry. This analysis revealed Arg 143 as the site of modification in MutY. Arg 143 and nearby Arg 147 are conserved throughout the endo III-like subfamily. Replacement of Arg 143 and Arg 147 with alanine by site-directed mutagenesis reduces adenine glycosylase activity of MutY toward OG:A and G:A mispairs. In addition, the R143A and R147A enzymes exhibit a reduced affinity for duplexes containing the substrate analogue 2'-deoxy-2'-fluoroadenosine opposite OG and G. Modeling of MutY bound to DNA using an endonuclease III-DNA complex structure shows that these two conserved arginines are located within close proximity to the DNA backbone. The insight from mass spectrometry experiments combined with functional mutagenesis results indicate that these two amino acids in the [4Fe-4S]2+ cluster-containing subfamily play an important role in recognition of the damaged DNA substrate.     

The flexible loop of human FEN1 endonuclease is required for flap cleavage during DNA replication and repair

The conserved, structure-specific flap endonuclease FEN1 cleaves 5' DNA flaps that arise during replication or repair. To address in vivo mechanisms of flap cleavage, we developed a screen for human FEN1 mutants that are toxic when expressed in yeast. Two targets were revealed: the flexible loop domain and the catalytic site. Toxic mutants caused G(2) arrest and cell death and were unable to repair methyl methanesulfonate lesions. All the mutant proteins retained flap binding. Unlike the catalytic site mutants, which lacked cleavage of any 5' flaps, the loop mutants exhibited partial ability to cut 5' flaps when an adjacent single nucleotide 3' flap was present. We suggest that the flexible loop is important for efficient cleavage through positioning the 5' flap and the catalytic site.     

Mycobacterium tuberculosis RecA intein possesses a novel ATP-dependent site-specific double-stranded DNA endonuclease activity

Mycobacterium tuberculosis recA harbors an intervening sequence in its open reading frame, presumed to encode an endonuclease (PI-MtuI) required for intein homing in inteinless recA allele. Although the protein-splicing ability of PI-MtuI has been characterized, the identification of its putative endonuclease activity has remained elusive. To investigate whether PI-MtuI possesses endonuclease activity, recA intervening sequence was cloned, overexpressed, and purified to homogeneity. Here we show that PI-MtuI bound both single- and double-stranded DNA with similar affinity but failed to cleave DNA in the absence of cofactors. Significantly, PI-MtuI nicked supercoiled DNA in the presence of alternative cofactors but required both Mn(2+) and ATP to generate linear double-stranded DNA. We observed that PI-MtuI was able to inflict a staggered double-strand break 24 bp upstream of the insertion site in the inteinless recA allele. Similar to a few homing endonucleases, DNA cleavage by PI-MtuI was specific with an exceptionally long cleavage site spanning 22 bp. The kinetic mechanism of PI-MtuI promoted cleavage supports a sequential rather than concerted pathway of strand cleavage with the formation of nicked double-stranded DNA as an intermediate. Together, these results reveal that RecA intein is a novel Mn(2+)-ATP-dependent double-strand specific endonuclease, which is likely to be important for homing process in vivo.     

Identification of a novel motif in DNA ligases exemplified by DNA ligase IV

DNA ligase IV is an essential protein that functions in DNA non-homologous end-joining, the major mechanism that rejoins DNA double-strand breaks in mammalian cells. LIG4 syndrome represents a human disorder caused by mutations in DNA ligase IV that lead to impaired but not ablated activity. Thus far, five conserved motifs in DNA ligases have been identified. We previously reported G469E as a mutational change in a LIG4 syndrome patient. G469 does not lie in any of the previously reported motifs. A sequence comparison between DNA ligases led us to identify residues 468-476 of DNA ligase IV as a further conserved motif, designated motif Va, present in eukaryotic DNA ligases. We carried out mutational analysis of residues within motif Va examining the impact on adenylation, double-stranded ligation, and DNA binding. We interpret our results using the DNA ligase I:DNA crystal structure. Substitution of the glycine at position 468 with an alanine or glutamic acid severely compromises protein activity and stability. Substitution of G469 with an alanine or glutamic acid is better tolerated but still impacts upon activity and protein stability. These finding suggest that G468 and G469 are important for protein stability and provide insight into the hypomorphic nature of the G469E mutation identified in a LIG4 syndrome patient. In contrast, residues 470, 473 and 476 within motif Va can be changed to alanine residues without any impact on DNA binding or adenylation activity. Importantly, however, such mutational changes do impact upon double-stranded ligation activity. Considered in light of the DNA ligase I:DNA crystal structure, our findings suggest that residues 470-476 function as part of a molecular pincer that maintains the DNA in a conformation that is required for ligation.     

Recognition of the oxidized lesions spiroiminodihydantoin and guanidinohydantoin in DNA by the mammalian base excision repair glycosylases NEIL1 and NEIL2

8-Oxoguanine (8-oxoG) is an unstable mutagenic DNA lesion that is prone to further oxidation. High valent metals such as Cr(V) and Ir(IV) readily oxidize 8-oxoG to form guanidinohydantoin (Gh), its isomer iminoallantoin (Ia), and spiroiminodihydantoin (Sp). When present in DNA, these lesions show enhanced base misincorporation over the parent 8-oxoG lesion leading to G --> T and G --> C transversion mutations and polymerase arrest. These findings suggested that further oxidized lesions of 8-oxoG are more mutagenic and toxic than 8-oxoG itself. Repair of oxidatively damaged bases, including Sp and Gh/Ia, are initiated by the base excision repair (BER) system that involves the DNA glycosylases Fpg, Nei, and Nth in E. coli. Mammalian homologs of two of these BER enzymes, OGG1 and NTH1, have little or no affinity for Gh/Ia and Sp. Herein we report that two recently identified mammalian glycosylases, NEIL1 and NEIL2, showed a high affinity for recognition and cleavage of DNA containing Gh/Ia and Sp lesions. NEIL1 and NEIL2 recognized both of these lesions in single-stranded DNA and catalyzed the removal of the lesions through a beta- and delta-elimination mechanism. NEIL1 and NEIL2 also recognized and excised the Gh/Ia lesion opposite all four natural bases in double-stranded DNA. NEIL1 was able to excise the Sp lesion opposite the four natural bases in double-stranded DNA, however, NEIL2 showed little cleavage activity against the Sp lesion in duplex DNA although DNA trapping studies show recognition and binding of NEIL2 to this lesion. This work suggests that NEIL1 and NEIL2 are essential in the recognition of further oxidized lesions arising from 8-oxoG and implies that these BER glycosylases may play an important role in the repair of DNA damage induced by carcinogenic metals.     

Endonucleolytic function of MutLalpha in human mismatch repair

Half of hereditary nonpolyposis colon cancer kindreds harbor mutations that inactivate MutLalpha (MLH1*PMS2 heterodimer). MutLalpha is required for mismatch repair, but its function in this process is unclear. We show that human MutLalpha is a latent endonuclease that is activated in a mismatch-, MutSalpha-, RFC-, PCNA-, and ATP-dependent manner. Incision of a nicked mismatch-containing DNA heteroduplex by this four-protein system is strongly biased to the nicked strand. A mismatch-containing DNA segment spanned by two strand breaks is removed by the 5'-to-3' activity of MutSalpha-activated exonuclease I. The probable endonuclease active site has been localized to a PMS2 DQHA(X)(2)E(X)(4)E motif. This motif is conserved in eukaryotic PMS2 homologs and in MutL proteins from a number of bacterial species but is lacking in MutL proteins from bacteria that rely on d(GATC) methylation for strand discrimination in mismatch repair. Therefore, the mode of excision initiation may differ in these organisms.