Summary DNA methylation is a major epigenetic mechanism for gene silencing. While methyltransferases mediate cytosine methylation, it is less clear how unmethylated regions in mammalian genomes are protected from de novo methylation and whether an active demethylating activity is involved. Here we show that either knockout or catalytic inactivation of the DNA repair enzyme Thymine DNA Glycosylase (TDG) leads to embryonic lethality in mice. TDG is necessary for recruiting p300 to retinoic acid (RA)-regulated promoters, protection of CpG islands from hypermethylation, and active demethylation of tissue-specific, developmentally- and hormonally-regulated promoters and enhancers. TDG interacts with the deaminase AID and the damage-response protein GADD45a. These findings highlight a dual role for TDG in promoting proper epigenetic states during development and suggest a two-step mechanism for DNA demethylation in mammals, whereby 5-methylcytosine and 5-hydroxymethylcytosine are first deaminated by AID to thymine and 5-hydroxymethyluracil, respectively, followed by TDG-mediated thymine and 5-hydroxymethyluracil excision repair.

Eukaryotic DNA polymerase beta (pol beta) can catalyze DNA synthesis during base excision DNA repair. It is shown here that pol beta also catalyzes release of 5'-terminal deoxyribose phosphate (dRP) residues from incised apurinic-apyrimidinic sites, which are common intermediate products in base excision repair. The catalytic domain for this activity resides within an amino-terminal 8-kilodalton fragment of pol beta, which comprises a distinct structural domain of the enzyme. Magnesium is required for the release of dRP from double-stranded DNA but not from a single-stranded oligonucleotide. Analysis of the released products indicates that the excision reaction occurs by beta-elimination rather than hydrolysis.

DNA ligase I belongs to a family of proteins that bind to proliferating cell nuclear antigen (PCNA) via a conserved 8-amino-acid motif [1]. Here we examine the biological significance of this interaction. Inactivation of the PCNA-binding site of DNA ligase I had no effect on its catalytic activity or its interaction with DNA polymerase beta. In contrast, the loss of PCNA binding severely compromised the ability of DNA ligase I to join Okazaki fragments. Thus, the interaction between PCNA and DNA ligase I is not only critical for the subnuclear targeting of the ligase, but also for coordination of the molecular transactions that occur during lagging-strand synthesis. A functional PCNA-binding site was also required for the ligase to complement hypersensitivity of the DNA ligase I mutant cell line 46BR.1G1 to monofunctional alkylating agents, indicating that a cytotoxic lesion is repaired by a PCNA-dependent DNA repair pathway. Extracts from 46BR.1G1 cells were defective in long-patch, but not short-patch, base-excision repair (BER). Our results show that the interaction between PCNA and DNA ligase I has a key role in long-patch BER and provide the first evidence for the biological significance of this repair mechanism.

DNA damage frequently leads to the production of apurinic/apyrimidinic (AP) sites, which are presumed to be repaired through the base excision pathway. For detailed analyses of this repair mechanism, a synthetic analog of an AP site, 3-hydroxy-2-hydroxymethyltetrahydrofuran (tetrahydrofuran), has been employed in a model system. Tetrahydrofuran residues are efficiently repaired in a Xenopus laevis oocyte extract in which most repair events involve ATP-dependent incorporation of no more than four nucleotides (Y. Matsumoto and D. F. Bogenhagen, Mol. Cell. Biol. 9:3750-3757, 1989; Y. Matsumoto and D. F. Bogenhagen, Mol. Cell. Biol. 11:44414447, 1991). Using a series of column chromatography procedures to fractionate X. laevis ovarian extracts, we developed a reconstituted system of tetrahydrofuran repair with five fractions, three of which were purified to near homogeneity: proliferating cell nuclear antigen (PCNA), AP endonuclease, and DNA polymerase 8. This PCNA-dependent system repaired natural AP sites as well as tetrahydrofuran residues. DNA polymerase ,I was able to replace DNA polymerase b only for repair of natural AP sites in a reaction that did not require PCNA. DNA polymerase a did not support repair of either type of AP site. This result indicates that AP sites can be repaired by two distinct pathways, the PCNA-dependent pathway and the DNA polymerase ,8-dependent pathway.Base excision repair is a major pathway for repair of damaged bases in DNA (8). In this pathway, a DNA-Nglycosylase initiates repair by removing a specifically modified base, leaving an apurinic/apyrimidinic (AP) site. AP sites are also generated by spontaneous or induced base loss. Since AP sites arise so frequently, it is reasonable to expect that cells should have evolved very efficient mechanisms to repair this sort of damage.To study the mechanism of repair of AP sites, we have taken advantage of the ability to insert a single copy of a synthetic analog of an AP site, 3-hydroxy-2-hydroxymethyltetrahydrofuran (tetrahydrofuran), at a specific position in a covalently closed circular DNA (cccDNA) (17,23,29 (215) 728-4333. ever, several lines of evidence suggested that DNA polymerase ,B might not be the only polymerase involved in repair of AP sites. First, our early experiments showed that repair of the tetrahydrofuran residue was inhibited by aphidicolin, as expected if repair involves a high-molecular-weight DNA polymerase (unpublished data). Second, biochemical analyses of yeast mutants indicated that DNA polymerase E is responsible for this reaction (33). While a discrepancy concerning the identity of the repair polymerase may be the result of intrinsic differences in the experimental systems, some uncertainties could be eliminated if a reconstituted system which catalyzes the repair reaction with purified factors were available. In this paper, we present results obtained by fractionating the X. laevis ovarian extract, leading to development of a reconstituted system. This system consists of five fractions, including proli...

The DNA mismatch repair (MMR) is a specialized system, highly conserved throughout evolution, involved in the maintenance of genomic integrity. To identify novel human genes that may function in MMR, we employed the yeast interaction trap. Using the MMR protein MLH1 as bait, we cloned MED1. The MED1 protein forms a complex with MLH1, binds to methyl-CpG-containing DNA, has homology to bacterial DNA repair glycosylases͞lyases, and displays endonuclease activity. Transfection of a MED1 mutant lacking the methyl-CpG-binding domain (MBD) is associated with microsatellite instability (MSI). These findings suggest that MED1 is a novel human DNA repair protein that may be involved in MMR and, as such, may be a candidate eukaryotic homologue of the bacterial MMR endonuclease, MutH. In addition, these results suggest that cytosine methylation may play a role in human DNA repair.

No abstract

Base excision repair can proceed in either one of two alternative pathways: a DNA polymerase ␤-dependent pathway and a proliferating cell nuclear antigen (PCNA)-dependent pathway. Excision of an apurinic/apyrimidinic (AP) site by cutting the phosphate backbone on its 3 side following incision at its 5 side by AP endonuclease is a prerequisite to completion of these repair pathways. Using a reconstituted system with the proteins derived from Xenopus laevis, we found that flap endonuclease 1 (FEN1) was a factor responsible for the excision of a 5-incised AP site in the PCNA-dependent pathway. In this pathway, DNA synthesis was not required for the action of FEN1 in the presence of PCNA and a replication factor C-containing fraction. The polymerase ␤-dependent pathway could also use FEN1 for excision of the synthetic AP sites, which were not susceptible to ␤-elimination. In this pathway, FEN1 was functional without PCNA and replication factor C but required the DNA synthesis, which led to a flap structure formation.Base excision repair is a major mechanism for repair of damaged bases in DNA (1). Major lesions to be repaired by this mechanism are bases with a relatively small modification or apurinic/apyrimidinic (AP) 1 sites. These types of damage are induced by ionizing radiation and exposure to various chemicals, such as alkylating agents, as well as by attack from reactive species generated during normal cellular metabolism. To protect their DNA from such persistent damage formation, most living organisms are equipped with vital mechanisms for base excision repair. In a present model for base excision repair (2), an altered base is removed by a specific DNA-N-glycosylase to leave an AP site, which is accordingly a common intermediate. The next step in this mechanism is the incision of the phosphate backbone immediately 5Ј to the AP site by AP endonuclease, generating a 3Ј-hydroxyl terminus and a 5Ј-terminus with a deoxyribose phosphate (dRP) group. The 5Ј-dRP residue is then excised, and a DNA polymerase fills in the gap. Finally, the DNA strand is sealed by the action of a DNA ligase.Recent studies with in vitro repair systems derived from vertebrates indicate that AP site repair in higher eukaryotes may proceed by either one of two alternative pathways: a DNA polymerase ␤ (pol ␤)-dependent pathway and a proliferating cell nuclear antigen (PCNA)-dependent pathway (3, 4). The pol ␤-dependent pathway requires a minimum of three proteins for AP site repair: AP endonuclease, pol ␤, and DNA ligase. In this pathway, pol ␤ catalyzes not only DNA synthesis but also excision of a dRP residue (5). This dRP excision is via ␤-elimination catalyzed by the amino-terminal 8-kDa domain of pol ␤. Consequently, the pol ␤-dependent pathway can repair an unmodified natural AP site efficiently but not a reduced AP site or a synthetic AP site analog, 3-hydroxy-2-hydroxymethyltetrahydrofuran (tetrahydrofuran

The human protein MED1 (also known as MBD4) was previously isolated in a two-hybrid screening using the mismatch repair protein MLH1 as a bait, and shown to have homology to bacterial base excision repair DNA N-glycosylases/lyases. To define the mechanisms of action of MED1, we implemented a sensitive glycosylase assay amenable to kinetic analysis. We show that MED1 functions as a mismatch-specific DNA N-glycosylase active on thymine, uracil, and 5-fluorouracil when these bases are opposite to guanine. MED1 lacks uracil glycosylase activity on single-strand DNA and abasic site lyase activity. The glycosylase activity of MED1 prefers substrates containing a G:T mismatch within methylated or unmethylated CpG sites; since G:T mismatches can originate via deamination of 5-methylcytosine to thymine, MED1 may act as a caretaker of genomic fidelity at CpG sites. A kinetic analysis revealed that MED1 displays a fast first cleavage reaction followed by slower subsequent reactions, resulting in biphasic time course; this is due to the tight binding of MED1 to the abasic site reaction product rather than a consequence of enzyme inactivation. Comparison of kinetic profiles revealed that the MED1 5-methylcytosine binding domain and methylation of the mismatched CpG site are not required for efficient catalysis.The integrity of genetic information is constantly challenged by a variety of endogenous and exogenous DNA damaging agents (1-3). Cellular DNA transactions occur in aqueous solution containing reactive oxygen species, and, as such, DNA is prone to both hydrolytic and oxidative damage. Hydrolysis of the N-glycosyl bond yields apurinic and, less frequently, apyrimidinic sites that are highly mutagenic. Hydrolytic deamination of cytosine and 5-methylcytosine (M) 1 generates G:U and G:T mismatches, respectively. Oxidative lesions include 8-oxoguanine, thymine glycol, and formamidopyrimidine derivatives of adenine and guanine (1, 2). In addition to endogenous damaging processes, DNA is exposed to the attack of exogenous reactive species, including alkylating agents and the carcinogens vinyl chloride and ethyl carbamate. Alkylating agents primarily alkylate the N 3 position of purines and the N 7 and O 6 positions of guanine (1, 2), whereas metabolites of vinyl chloride and ethyl carbamate generate cyclic (etheno) DNA adducts, such as 3,N 4 -ethenocytosine, 1,N 6 -ethenoadenine, 1,N 2 -ethenoguanine and N 2 ,3-ethenoguanine (4, 5). Efficient correction of these DNA lesions relies on the action of several enzymes belonging to the base excision repair system (2, 6 -9). Unlike nucleotide excision repair or long-patch mismatch repair (MMR), base excision repair enzymes usually act in a lesion-specific fashion on a single damaged or mismatched nucleotide. Given the mutagenic potential of DNA lesions, continuing elucidation of the biochemical activities, damage spectrum and specificity of base excision repair enzymes has direct implications on cancer and aging (2).In an effort to isolate new human proteins involved in DNA repair, ...