Christopher L. Millington

Flipping of alkylated DNA damage bridges base and nucleotide excision repair

Alkyltransferase-like proteins (ATLs) share functional motifs with the cancer chemotherapy target O6-alkylguanine-DNA alkyltransferase (AGT) and paradoxically protect cells from the biological effects of DNA alkylation damage, despite lacking the reactive cysteine and alkyltransferase activity of AGT. Here we determine Schizosaccharomyces pombe ATL structures without and with damaged DNA containing the endogenous lesion O6-methylguanine or cigarette-smoke-derived O6-4-(3-pyridyl)-4-oxobutylguanine. These results reveal non-enzymatic DNA nucleotide flipping plus increased DNA distortion and binding pocket size compared to AGT. Our analysis of lesion-binding site conservation identifies new ATLs in sea anemone and ancestral archaea, indicating that ATL interactions are ancestral to present-day repair pathways in all domains of life. Genetic connections to mammalian XPG (also known as ERCC5) and ERCC1 in S. pombe homologues Rad13 and Swi10 and biochemical interactions with Escherichia coli UvrA and UvrC combined with structural results reveal that ATLs sculpt alkylated DNA to create a genetic and structural intersection of base damage processing with nucleotide excision repair.

Atl1 Regulates Choice between Global Genome and Transcription-Coupled Repair of O(6)-Alkylguanines.

Nucleotide excision repair (NER) has long been known to remove DNA lesions induced by chemical carcinogens, and the molecular mechanism has been partially elucidated. Here we demonstrate that in Schizosaccharomyces pombe a DNA recognition protein, alkyltransferase-like 1 (Atl1), can play a pivotal role in selecting a specific NER pathway, depending on the nature of the DNA modification. The relative ease of dissociation of Atl1 from DNA containing small O(6)-alkylguanines allows accurate completion of global genome repair (GGR), whereas strong Atl1 binding to bulky O(6)-alkylguanines blocks GGR, stalls the transcription machinery, and diverts the damage to transcription-coupled repair. Our findings redraw the initial stages of the NER process in those organisms that express an alkyltransferase-like gene and raise the question of whether or not O(6)-alkylguanine lesions that are poor substrates for the alkyltransferase proteins in higher eukaryotes might, by analogy, signal such lesions for repair by NER.

The nitrosated bile acid DNA lesion O6-carboxymethylguanine is a substrate for the human DNA repair protein O6-methylguanine-DNA methyltransferase

The consumption of red meat is a risk factor in human colorectal cancer (CRC). One hypothesis is that red meat facilitates the nitrosation of bile acid conjugates and amino acids, which rapidly convert to DNA-damaging carcinogens. Indeed, the toxic and mutagenic DNA adduct O6-carboxymethylguanine (O6-CMG) is frequently present in human DNA, increases in abundance in people with high levels of dietary red meat and may therefore be a causative factor in CRC. Previous reports suggested that O6-CMG is not a substrate for the human version of the DNA damage reversal protein O6-methylguanine-DNA methyltransferase (MGMT), which protects against the genotoxic effects of other O6-alkylguanine lesions by removing alkyl groups from the O6-position. We now show that synthetic oligodeoxyribonucleotides containing the known MGMT substrate O6-methylguanine (O6-MeG) or O6-CMG effectively inactivate MGMT in vitro (IC50 0.93 and 1.8 nM, respectively). Inactivation involves the removal of the O6-alkyl group and its transfer to the active-site cysteine residue of MGMT. O6-CMG is therefore an MGMT substrate, and hence MGMT is likely to be a protective factor in CRC under conditions where O6-CMG is a potential causative agent.

Alkyltransferase-like protein (Atl1) distinguishes alkylated guanines for DNA repair using cation–π interactions

Alkyltransferase-like (ATL) proteins in Schizosaccharomyces pombe (Atl1) and Thermus thermophilus (TTHA1564) protect against the adverse effects of DNA alkylation damage by flagging O6-alkylguanine lesions for nucleotide excision repair (NER). We show that both ATL proteins bind with high affinity to oligodeoxyribonucleotides containing O6-alkylguanines differing in size, polarity, and charge of the alkyl group. However, Atl1 shows a greater ability than TTHA1564 to distinguish between O6-alkylguanine and guanine and in an unprecedented mechanism uses Arg69 to probe the electrostatic potential surface of O6-alkylguanine, as determined using molecular mechanics calculations. An unexpected consequence of this feature is the recognition of 2,6-diaminopurine and 2-aminopurine, as confirmed in crystal structures of respective Atl1-DNA complexes. O6-Alkylguanine and guanine discrimination is diminished for Atl1 R69A and R69F mutants, and S. pombe R69A and R69F mutants are more sensitive toward alkylating agent toxicity, revealing the key role of Arg69 in identifying O6-alkylguanines critical for NER recognition.

Insights into the DNA stabilizing contributions of a bicyclic cytosine analogue: crystal structures of DNA duplexes containing 7,8-dihydropyrido [2,3-d]pyrimidin-2-one

The incorporation of the bicyclic cytosine analogue 7,8-dihydropyrido[2,3-d]pyrimidin-2-one (X) into DNA duplexes results in a significant enhancement of their stability (3–4 K per modification). To establish the effects of X on the local hydrogen-bonding and base stacking interactions and the overall DNA conformation, and to obtain insights into the correlation between the structure and stability of X-containing DNA duplexes, the crystal structures of [d(CGCGAATT-X-GCG)]2 and [d(CGCGAAT-X-CGCG)]2 have been determined at 1.9–2.9 Å resolutions. In all of the structures, the analogue X base pairs with the purine bases on the opposite strands through Watson–Crick and/or wobble type hydrogen bonds. The additional ring of the X base is stacked on the thymine bases at the 5′-side and overall exhibits greatly enhanced stacking interactions suggesting that this is a major contribution to duplex stabilization.

Structures of DNA duplexes containing O6-carboxymethylguanine, a lesion associated with gastrointestinal cancer, reveal a mechanism for inducing pyrimidine transition mutations

N-nitrosation of glycine and its derivatives generates potent alkylating agents that can lead to the formation of O6-carboxymethylguanine (O6-CMG) in DNA. O6-CMG has been identified in DNA derived from human colon tissue, and its occurrence has been linked to diets high in red and processed meats. By analogy to O6-methylguanine, O6-CMG is expected to be highly mutagenic, inducing G to A mutations during DNA replication that can increase the risk of gastrointestinal and other cancers. Two crystal structures of DNA dodecamers d(CGCG[O6-CMG]ATTCGCG) and d(CGC[O6-CMG]AATTCGCG) in complex with Hoechst33258 reveal that each can form a self-complementary duplex to retain the B-form conformation. Electron density maps clearly show that O6-CMG forms a Watson–Crick–type pair with thymine similar to the canonical A:T pair, and it forms a reversed wobble pair with cytosine. In situ structural modeling suggests that a DNA polymerase can accept the Watson–Crick–type pair of O6-CMG with thymine, but might also accept the reversed wobble pair of O6-CMG with cytosine. Thus, O6-CMG would permit the mis-incorporation of dTTP during DNA replication. Alternatively, the triphosphate that would be formed by carboxymethylation of the nucleotide triphosphate pool d[O6-CMG]TP might compete with dATP incorporation opposite thymine in a DNA template.

Bypass of Mutagenic O6-Carboxymethylguanine DNA Adducts by Human Y- and B-Family Polymerases

The generation of chemical alkylating agents from nitrosation of glycine and bile acid conjugates in the gastrointestinal tract is hypothesized to initiate carcinogenesis. O(6)-carboxymethylguanine (O(6)-CMG) is a product of DNA alkylation derived from nitrosated glycine. Although the tendency of the structurally related adduct O(6)-methylguanine to code for the misincoporation of TTP during DNA replication is well-established, the impact of the presence of the O(6)-CMG adduct in a DNA template on the efficiency and fidelity of translesion DNA synthesis (TLS) by human DNA polymerases (Pols) has hitherto not been described. Herein, we characterize the ability of the four human TLS Pols η, ι, κ, and ζ and the replicative Pol δ to bypass O(6)-CMG in a prevalent mutational hot-spot for colon cancer. The results indicate that Pol η replicates past O(6)-CMG, incorporating dCMP or dAMP, whereas Pol κ incorporates dCMP only, and Pol ι incorporates primarily dTMP. Additionally, the subsequent extension step was carried out with high efficiency by TLS Pols η, κ, and ζ, while Pol ι was unable to extend from a terminal mismatch. These results provide a first basis of O(6)-CMG-promoted base misincorporation by Y- and B-family polymerases potentially leading to mutational signatures associated with colon cancer.

O 6-Carboxymethylguanine in DNA forms a sequence context-dependent wobble base-pair structure with thymine

N-Nitrosation of glycine and its derivatives generates potent alkylating agents that can lead to the formation of O(6)-carboxymethylguanine (O(6)-CMG) in DNA. O(6)-CMG has been identified in DNA derived from human colon tissue and its occurrence has been linked to diets high in red and processed meats, implying an association with the induction of colorectal cancer. By analogy to O(6)-methylguanine, O(6)-CMG is expected to be mutagenic, inducing G-to-A mutations that may be the molecular basis of increased cancer risk. Previously, the crystal structure of the DNA dodecamer d(CGCG[O(6)-CMG]ATTCGCG) has been reported, in which O(6)-CMG forms a Watson-Crick-type pair with thymine similar to the canonical A:T pair. In order to further investigate the versatility of O(6)-CMG in base-pair formation, the structure of the DNA dodecamer d(CGC[O(6)-CMG]AATTTGCG) containing O(6)-CMG at a different position has been determined by X-ray crystallography using four crystal forms obtained under conditions containing different solvent ions (Sr(2+), Ba(2+), Mg(2+), K(+) or Na(+)) with and without Hoechst 33258. The most striking finding is that the pairing modes of O(6)-CMG with T are quite different from those previously reported. In the present dodecamer, the T bases are displaced (wobbled) into the major groove to form a hydrogen bond between the thymine N(3) N-H and the carboxyl group of O(6)-CMG. In addition, a water molecule is bridged through two hydrogen bonds between the thymine O(2) atom and the 2-amino group of O(6)-CMG to stabilize the pairing. These interaction modes commonly occur in the four crystal forms, regardless of the differences in crystallization conditions. The previous and the present results show that O(6)-CMG can form a base pair with T in two alternative modes: the Watson-Crick type and a high-wobble type, the nature of which may depend on the DNA-sequence context.

Crystal structures of DNA duplexes stabilized by bicyclic-C residues.

Chemical modification of nucleic acids is being studied extensively as an approach for the development of nucleic acid-based therapies. We found that a nucleotide carrying 7,8-dihydropyrido[2,3-d]pyrimidin-2-one (bicyclic-C or X), which is a cytosine derivative with a propene attached at the N4 and C5 atoms, increases the stability of DNA duplexes. To establish the conformational effects of X on DNA and to obtain insight into the correlation between the structure and stability of X-containing DNA duplexes, the crystal structures of [d(CGCGAATT-X-GCG)](2) and [d(CGCGAAT-X-CGCG)](2) have been determined at 2.9 A resolutions. In both duplexes, the bicyclic-C bases form pairs with the counter bases through hydrogen bonds, and stabilize the duplex formation in part by stacking interactions between X and the subsequent thymine base of the same strand.