Any uracil bases in DNA, a result of either misincorporation or deamination of cytosine, are removed by uracil-DNA glycosylase (UDG), one of the most efficient and specific of the base-excision DNA-repair enzymes. Crystal structures of human and viral UDGs complexed with free uracil have indicated that the enzyme binds an extrahelical uracil. Such binding of undamaged extrahelical bases has been seen in the structures of two bacterial methyltransferases and bacteriophage T4 endonuclease V. Here we characterize the DNA binding and kinetics of several engineered human UDG mutants and present the crystal structure of one of these, which to our knowledge represents the first structure of any eukaryotic DNA repair enzyme in complex with its damaged, target DNA. Electrostatic orientation along the UDG active site, insertion of an amino acid (residue 272) into the DNA through the minor groove, and compression of the DNA backbone flanking the uracil all result in the flipping-out of the damaged base from the DNA major groove, allowing specific recognition of its phosphate, deoxyribose and uracil moieties. Our structure thus provides a view of a productive complex specific for cleavage of uracil from DNA and also reveals the basis for the enzyme-assisted nucleotide flipping by this critical DNA-repair enzyme.

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Activation-induced cytidine deaminase (AID) is a 'master molecule' in immunoglobulin (Ig) class-switch recombination (CSR) and somatic hypermutation (SHM) generation, AID deficiencies are associated with hyper-IgM phenotypes in humans and mice. We show here that recessive mutations of the gene encoding uracil-DNA glycosylase (UNG) are associated with profound impairment in CSR at a DNA precleavage step and with a partial disturbance of the SHM pattern in three patients with hyper-IgM syndrome. Together with the finding that nuclear UNG expression was induced in activated B cells, these data support a model of CSR and SHM in which AID deaminates cytosine into uracil in targeted DNA (immunoglobulin switch or variable regions), followed by uracil removal by UNG.

hUNG2 and hSMUG1 are the only known glycosylases that may remove uracil from both double-and singlestranded DNA in nuclear chromatin, but their relative contribution to base excision repair remains elusive. The present study demonstrates that both enzymes are strongly stimulated by physiological concentrations of Mg 2؉, at which the activity of hUNG2 is 2-3 orders of magnitude higher than of hSMUG1. Moreover, Mg 2؉ increases the preference of hUNG2 toward uracil in ssDNA nearly 40-fold. APE1 has a strong stimulatory effect on hSMUG1 against dsU, apparently because of enhanced dissociation of hSMUG1 from AP sites in dsDNA. hSMUG1 also has a broader substrate specificity than hUNG2, including 5-hydroxymethyluracil and 3,N 4 -ethenocytosine. hUNG2 is excluded from, whereas hSMUG1 accumulates in, nucleoli in living cells. In contrast, only hUNG2 accumulates in replication foci in the S-phase. hUNG2 in nuclear extracts initiates base excision repair of plasmids containing either U:A and U:G in vitro. Moreover, an additional but delayed repair of the U:G plasmid is observed that is not inhibited by neutralizing antibodies against hUNG2 or hSMUG1. We propose a model in which hUNG2 is responsible for both prereplicative removal of deaminated cytosine and postreplicative removal of misincorporated uracil at the replication fork. We also provide evidence that hUNG2 is the major enzyme for removal of deaminated cytosine outside of replication foci, with hSMUG1 acting as a broad specificity backup.Uracil in DNA can be introduced via two mechanisms, deamination of cytosine and misincorporation of dUMP during replication. Deamination of cytosine has been calculated from measured deamination rates to occur at a rate of 100 -500 per human cell/day (1, 2) to yield mutagenic U:G mispairs. Uracil may also appear as a consequence of misincorporation of dUMP instead of dTMP during replication, resulting in a U:A base pair. The latter is not miscoding, but may produce cytotoxic and mutagenic AP site intermediates during repair. In organisms containing 5-methylcytosine in their genomes, deamination of 5-methylcytosine furthermore leads to T:G mismatches. All living organisms express uracil-DNA glycosylases (UDGs) 1 that prevent cytotoxic and mutagenic effects of the above lesions. UDGs remove uracil (and sometimes other damaged bases or thymine) from the deoxyribose and thus initiate a multistep base excision repair (BER) pathway, eventually restoring the correct DNA sequence. After removal of uracil by an UDG and cleavage of the resulting abasic site by AP endonuclease (APE1/APE2), the BER pathway splits into two branches (reviewed in Ref.3). The presumed major track is the shortpatch pathway. It uses the 5Ј-deoxyribophosphodiesterase activity of DNA polymerase ␤ to cleave 3Ј of the abasic site, thus releasing deoxyribose-5-phosphate. Then pol ␤ inserts C or T, depending on the template base. Finally, DNA ligase III seals the nick, perhaps aided by the scaffold protein XRCC1. The alternative long-patch pathway largely uses replicatio...

Crystal structures of the DNA repair enzyme human uracil-DNA glycosylase (UDG), combined with mutational analysis, reveal the structural basis for the specificity of the enzyme. Within the classic alpha/beta fold of UDG, sequence-conserved residues form a positively charged, active-site groove the width of duplex DNA, at the C-terminal edge of the central four-stranded parallel beta sheet. In the UDG-6-aminouracil complex, uracil binds at the base of the groove within a rigid preformed pocket that confers selectivity for uracil over other bases by shape complementary and by main chain and Asn-204 side chain hydrogen bonds. Main chain nitrogen atoms are positioned to stabilize the oxyanion intermediate generated by His-268 acting via nucleophilic attack or general base mechanisms. Specific binding of uracil flipped out from a DNA duplex provides a structural mechanism for damaged base recognition.