Detailed analysis of in-hospital transmission of SARS-CoV-2 using whole genome sequencing.

BACKGROUND: Hospital transmission of SARS-CoV-2 has proved difficult to control, with healthcare-associated infections troublesome throughout. AIM: To understand factors contributing to hospital transmission of infections, which is necessary for containing spread. METHODS: An outbreak of 56 staff and patient cases of COVID-19 over a 31-day period in a tertiary referral unit is presented, with at least a further 29 cases identified outside of the unit and the hospital by whole genome sequencing (WGS). FINDINGS: Transmission is documented from staff to staff, staff to patients, and patients to staff, showing disruption of a tertiary referral service, despite implementation of nationally recommended control measures, superior ventilation, and use of personal protective equipment. There was extensive spread from the index case, despite this patient spending only 10 h bed bound on the ward in strict cubicle isolation and with an initial single target low level (CT = 32) polymerase chain reaction test. CONCLUSION: This investigation highlights how effectively and rapidly SARS-CoV-2 can spread in certain circumstances. It raises questions about infection control measures in place at the time and calls into question the premise that transmissibility can be reliably detected by using lower sensitivity rapid antigen lateral flow tests. We also highlight the value of early intervention in reducing impact as well as the value of WGS in understanding outbreaks.

Zoonotic Transfer of Clostridium difficile Harboring Antimicrobial Resistance between Farm Animals and Humans.

The emergence of Clostridium difficile as a significant human diarrheal pathogen is associated with the production of highly transmissible spores and the acquisition of antimicrobial resistance genes (ARGs) and virulence factors. Unlike the hospital-associated C. difficile RT027 lineage, the community-associated C. difficile RT078 lineage is isolated from both humans and farm animals; however, the geographical population structure and transmission networks remain unknown. Here, we applied whole-genome phylogenetic analysis of 248 C. difficile RT078 strains from 22 countries. Our results demonstrate limited geographical clustering for C. difficile RT078 and extensive coclustering of human and animal strains, thereby revealing a highly linked intercontinental transmission network between humans and animals. Comparative whole-genome analysis reveals indistinguishable accessory genomes between human and animal strains and a variety of antimicrobial resistance genes in the pangenome of C. difficile RT078. Thus, bidirectional spread of C. difficile RT078 between farm animals and humans may represent an unappreciated route disseminating antimicrobial resistance genes between humans and animals. These results highlight the importance of the "One Health" concept to monitor infectious disease emergence and the dissemination of antimicrobial resistance genes.

Whole genome sequencing of Shigella sonnei through PulseNet Latin America and Caribbean: advancing global surveillance of foodborne illnesses.

OBJECTIVES: Shigella sonnei is a globally important diarrhoeal pathogen tracked through the surveillance network PulseNet Latin America and Caribbean (PNLA&C), which participates in PulseNet International. PNLA&C laboratories use common molecular techniques to track pathogens causing foodborne illness. We aimed to demonstrate the possibility and advantages of transitioning to whole genome sequencing (WGS) for surveillance within existing networks across a continent where S. sonnei is endemic. METHODS: We applied WGS to representative archive isolates of S. sonnei (n = 323) from laboratories in nine PNLA&C countries to generate a regional phylogenomic reference for S. sonnei and put this in the global context. We used this reference to contextualise 16 S. sonnei from three Argentinian outbreaks, using locally generated sequence data. Assembled genome sequences were used to predict antimicrobial resistance (AMR) phenotypes and identify AMR determinants. RESULTS: S. sonnei isolates clustered in five Latin American sublineages in the global phylogeny, with many (46%, 149 of 323) belonging to previously undescribed sublineages. Predicted multidrug resistance was common (77%, 249 of 323), and clinically relevant differences in AMR were found among sublineages. The regional overview showed that Argentinian outbreak isolates belonged to distinct sublineages and had different epidemiologic origins. CONCLUSIONS: Latin America contains novel genetic diversity of S. sonnei that is relevant on a global scale and commonly exhibits multidrug resistance. Retrospective passive surveillance with WGS has utility for informing treatment, identifying regionally epidemic sublineages and providing a framework for interpretation of prospective, locally sequenced outbreaks.

Whole genome sequencing reveals potential spread of Clostridium difficile between humans and farm animals in the Netherlands, 2002 to 2011.

Farm animals are a potential reservoir for human Clostridium difficile infection (CDI), particularly PCR ribotype 078 which is frequently found in animals and humans. Here, whole genome single-nucleotide polymorphism (SNP) analysis was used to study the evolutionary relatedness of C. difficile 078 isolated from humans and animals on Dutch pig farms. All sequenced genomes were surveyed for potential antimicrobial resistance determinants and linked to an antimicrobial resistance phenotype. We sequenced the whole genome of 65 C. difficile 078 isolates collected between 2002 and 2011 from pigs (n = 19), asymptomatic farmers (n = 15) and hospitalised patients (n = 31) in the Netherlands. The collection included 12 pairs of human and pig isolates from 2011 collected at 12 different pig farms. A mutation rate of 1.1 SNPs per genome per year was determined for C. difficile 078. Importantly, we demonstrate that farmers and pigs were colonised with identical (no SNP differences) and nearly identical (less than two SNP differences) C. difficile clones. Identical tetracycline and streptomycin resistance determinants were present in human and animal C. difficile 078 isolates. Our observation that farmers and pigs share identical C. difficile strains suggests transmission between these populations, although we cannot exclude the possibility of transmission from a common environmental source.

Distinguishable epidemics of multidrug-resistant Salmonella Typhimurium DT104 in different hosts.

The global epidemic of multidrug-resistant Salmonella Typhimurium DT104 provides an important example, both in terms of the agent and its resistance, of a widely disseminated zoonotic pathogen. Here, with an unprecedented national collection of isolates collected contemporaneously from humans and animals and including a sample of internationally derived isolates, we have used whole-genome sequencing to dissect the phylogenetic associations of the bacterium and its antimicrobial resistance genes through the course of an epidemic. Contrary to current tenets supporting a single homogeneous epidemic, we demonstrate that the bacterium and its resistance genes were largely maintained within animal and human populations separately and that there was limited transmission, in either direction. We also show considerable variation in the resistance profiles, in contrast to the largely stable bacterial core genome, which emphasizes the critical importance of integrated genotypic data sets in understanding the ecology of bacterial zoonoses and antimicrobial resistance.

Retrospective analysis of whole genome sequencing compared to prospective typing data in further informing the epidemiological investigation of an outbreak of Shigella sonnei in the UK.

The aim of this study was to retrospectively assess the value of whole genome sequencing (WGS) compared to conventional typing methods in the investigation and control of an outbreak of Shigella sonnei in the Orthodox Jewish (OJ) community in the UK. The genome sequence analysis showed that the strains implicated in the outbreak formed three phylogenetically distinct clusters. One cluster represented cases associated with recent exposure to a single strain, whereas the other two clusters represented related but distinct strains of S. sonnei circulating in the OJ community across the UK. The WGS data challenged the conclusions drawn during the initial outbreak investigation and allowed cases of dysentery to be implicated or ruled out of the outbreak that were previously misclassified. This study showed that the resolution achieved using WGS would have clearly defined the outbreak, thus facilitating the promotion of infection control measures within local schools and the dissemination of a stronger public health message to the community.

Excision by the human methylpurine DNA N-glycosylase of cyanuric acid, a stable and mutagenic oxidation product of 8-oxo-7,8-dihydroguanine.

PURPOSE: 1-(2-Deoxy-beta-D-erythro-pentofuranosyl)-cyanuric acid (cyanuric acid nucleoside or dCa) has been shown to be formed upon exposure of 8-oxo-7,8-dihydroguanine- (8-oxoG) containing oligodeoxyribonucleotides (ODN) to oxidizing agents. When present in DNA, cyanuric acid (Ca) is readily bypassed by Escherichia coli DNA polymerases, which preferentially incorporate 2'-deoxyadenosine-5'-monophosphate (dAMP) opposite to the lesion. Therefore, Ca could be a mutagenic DNA lesion yielding G.C to T.A transversions like 8-oxoG. These results call attention to the potential importance of secondary oxidation products of 8-oxoG. The present study investigates the capability of several DNA N-glycosylases to remove the Ca lesion in DNA. MATERIALS AND METHODS: A site-specifically modified 22-mer ODN containing a single Ca residue was hybridized with complementary sequences yielding four DNA duplexes harbouring Ca opposite each of the regular DNA bases. The four Ca.N duplexes were used as substrates for nine DNA N-glycosylases from bacterial, yeast or human origin. RESULTS: The results show that the human methylpurine DNA N-glycosylase (Mpg) can remove Ca from DNA duplexes. Interestingly, oxidized base-specific DNA N-glycosylases, Fpg, Nth, Ntg1, Ntg2, Ogg1, hNth1 and hOgg1, cannot repair Ca in DNA. Furthermore, the removal of Ca by Mpg varied markedly depending on the opposite DNA base, the rank being Ca.C=Ca.T>Ca.G=Ca.A. CONCLUSIONS: 8-OxoG-derived lesions in DNA such as spiroiminodihydantoin (Sp), guanidinohydantoin (Gh), oxaluric acid (Oa), oxazolone (Oz) and Ca are substrates of base excision repair DNA N-glycosylases. Most of them, Sp, Gh, Oa and Oz, are substrates of the oxidized bases-specific enzymes such as Nth or Fpg. In contrast, Ca is substrate of the human methylpurine DNA N-glycosylase (Mpg).

Ligation-mediated PCR: robotic liquid handling for DNA damage and repair.

The investigation of in vivo DNA repair in mammalian cells at nucleotide resolution requires the quantification of break frequencies less than one per kilobase. By optimizing several parameters of the ligation-mediated PCR technique, we find that the required sensitivity can be achieved. We also report details of a one-day procedure that can be performed either with or without a robotic liquid handling workstation. The use of near-infrared fluorescent-labeled primers with detection by a LI-COR DNA sequencer provides for safe, nonradioactive detection, similar in sensitivity to the use of 32P-labeled primers but with the additional advantage that high-quality digitized data are obtained directly. Multiplexing can be performed; that is, more than one sequence can be analyzed in a single reaction, and multiple reactions can be processed robotically. Primer sets for exons 5-8 of the tumor suppressor gene, p53, were designed for simultaneous thermal cycling. The improved procedure with infrared detection was used to monitor low-frequency damage (<1 break/kb) and/or repair of UVB, UVC, and chemical methylation. Quantitative data on the linearity of response and reproducibility are described. The coefficient of variation for technical replicates was typically 10%. The methods described here will permit high sample throughput for the detection of DNA damage and repair as well as in vivo protein footprints.

DNA glycosylase activity assay based on streptavidin paramagnetic bead substrate capture.

A method to detect DNA glycosylase activity is described. The substrate used was an oligodeoxyribonucleotide with a unique hypoxanthine base, but has general application to any DNA glycosylase or endonuclease. The oligodeoxyribonucleotide was labeled at the 5' end with 32P and at the 3' end with a biotin linkage and annealed to a complementary oligodeoxyribonucleotide. The hypoxanthine base was excised in solution using the MPG protein, a human DNA glycosylase. Following cleavage of the phosphodiester linkage by NaOH, the oligodeoxyribonucleotide was attached to streptavidin-coated, paramagnetic beads. Binding of the labeled oligodeoxyribonucleotide to the beads was indicative of the kinetics of the reaction. As a control, half of the reaction products were loaded on to a denaturing polyacrylamide gel. Comparable values for steady-state kinetic constants were obtained using both methods. This nonelectrophoretic technique is a rapid assay of DNA glycosylase activity for both purified proteins and crude extracts. This method can be directly adapted for high-throughput techniques.

Characterization and mapping of DNA damage induced by reactive metabolites of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) at nucleotide resolution in human genomic DNA.

The nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is an important tobacco-specific carcinogen associated with lung cancer. Its complex enzymatic activation, leading to methyl and pyridyloxobutyl (POB)-modified DNA, makes DNA damage difficult to characterize and quantify. Therefore, we use the NNK analogue 4-[(acetoxymethyl)nitrosamino]-1-(3-pyridyl)-1-butanone (NNKOAc) to induce damage in genomic DNA, and to map the sites and frequency of adducts at nucleotide resolution using ligation-mediated polymerase chain reaction and terminal transferase-dependent polymerase chain reactions (LMPCR and TDPCR). NNKOAc induced single-strand breaks in a concentration-dependent manner. Post-alkylation treatments, including hot piperidine or digestion with the enzymes Escherichia coli 3-methyladenine-DNA glycosylase II, formamidopyrimidine-DNA glycosylase, Escherichia coli endonuclease III, or phage T4 UV endonuclease V did not increase the level of DNA breaks in NNKOAc-treated DNA. Detection of DNA damage using LMPCR was possible only when POB-DNA was 5'-phosphorylated prior to the LMPCR procedure. NNKOAc generated damage at all four bases with the decreasing order guanine>adenine>cytosine>thymine. In contrast to NNKOAc damage distribution patterns, those induced by N-nitroso(acetoxymethyl)methylamine, a methylating NNK analog, induced damage principally at G positions detectable by enzymatic means that did not require phosphorylation. Analysis of damage distribution patterns, reveals a high frequency of damage in the p53 gene in codons 241 and 245 and a lower frequency of damage in codon 248. We analyzed the 3' termini of the NNKOAc induced single-strand breaks using a (32)P-post-labeling assay or a nucleotide exchange reaction at the 3'-termini catalyzed by T4 DNA polymerase combined with endonuclease IV treatment. Both methods indicate that the 3' termini of the single-strand breaks are not hydroxyl groups and are blocked by an unknown chemical structure that is not recognized by endonuclease IV. These data are consistent with POB-phosphotriester hydrolysis leading to strand breaks in DNA. The POB-damage could be mutagenic because NNKOAc produces single-strand breaks with the products being a 5'-hydroxyl group and a 3'-blocking group and strand breaks. These results represent the first step in determining if NNK pyridyloxobutylates DNA with sequence specificity similar to those observed with other model compounds.

Excision repair of adozelesin-N3 adenine adduct by 3-methyladenine-DNA glycosylases and UvrABC nuclease.

Adozelesin is a synthetic analog of the antitumor antibiotic CC-1065, which alkylates the N3 of adenine in the minor groove in a sequence-selective manner. Since the cytotoxic potency of a DNA alkylating agent can be modulated by DNA excision repair system, we investigated whether nucleotide excision repair (NER) and base excision repair (BER) enzymes are able to excise the bulky DNA adduct induced by adozelesin. The UvrABC nuclease and 3-methyladenine-DNA glycosylase, that exhibit a broad spectrum of substrate specificity, were selected as typical NER and BER enzymes, respectively. The adozelesin-DNA adduct was first formed in the radiolabeled restriction DNA fragment and its excision by purified repair enzymes was monitored on a DNA sequencing gel. The treatment of the DNA adduct with a purified UvrABC nuclease and sequencing gel analysis of cleaved DNA showed that UvrABC nuclease was able to incise the adozelesin adduct. The incision site corresponded to the general nuclease incision site. Excision of this adduct by 3-methyladenine-DNA glycosylases was determined following the treatment of the DNA adduct with a homogeneous recombinant bacterial, rat and human 3-methyladenine-DNA glycosylases. Abasic sites generated by DNA glycosyalses were cleaved by the associated lyase activity of the E. coli formamidopyrimidine-DNA glycosylase (Fpg). Resolution of cleaved DNA on a sequencing gel showed that the DNA glycosylase from different sources could not release the N3-adenine adducts. A cytotoxicity assay using E. coli repair mutant strains showed that E. coli mutant strains defective in the uvrA gene were more sensitive to cell killing by adozelesin than E. coli mutant strain defective in the alkA gene or the wild type. These results suggest that the NER pathway seems to be the major excision repair system in protecting cells from the cytotoxicity of adozelesin.

Alkylating agent and chromatin structure determine sequence context-dependent formation of alkylpurines.

We determined the adduct maps of S(N)1 and S(N)2 alkylating agents in cultured human cells (in vivo) and in vitro to probe DNA-protein interactions along sequences of the promoter and exon 1 of the Fragile-X mental retardation 1 (FMR1) gene. Using ligation-mediated polymerase chain reaction (LMPCR), we compared the piperidine-sensitive alkylpurines sites generated by treating cultured cells (in vivo) and naked DNA (in vitro) with S(N)1 (N-methyl-N-nitrosourea, N-nitroso(acetoxymethyl)methylamine and 1-methyl-3-nitro-1-nitrosoguanidine) and S(N)2 alkylating agents (dimethyl sulfate (DMS), methane sulfonic acid methyl ester, iodo methane, diethyl sulfate, methane sulfonic acid ethyl ester and iodo ethane). The FMR1 promoter has four sites where DNA-protein interactions are observed. In these regions, the S(N)1 methylating agent reactions produced only hypo-reactive sites. In contrast, iodoalkane S(N)2 alkylating agents (MeI and EtI) reactions generated only hyper-reactive sites. Although there are hyper-reactive sites for the other S(N)2 reagents, the hyper-reactive site at +14 on the FMR1 map is more pronounced for the sulfate and sulfonate-derived alkylating agents than for the iodoalkanes. However, DMS modification in the presence of methyl sulfone, a compound that does not alkylate DNA, eliminates the hyper-reactive site observed at +14. This suggests that the electron-rich oxygen atoms of the sulfate and sulfonate-derived S(N)2 alkylating agent structure position the alkylating moiety to the neighboring N-7-guanine position to favor alkyl transfer to the guanine. Using KMnO(4) to probe for single-strand DNA, an unpaired cytosine base was detected at the 5'-side of the hyper- reactive guanine base at position +14, consistent with the formation of a local DNA single-strand bulge. In conclusion, we show that the sequence context-dependent formation of alkylpurines is determined by the chemical nature of the alkylating agent, the DNA sequence context, chromatin structure, and the presence of other non-reactive molecules that can inhibit alkylation.

Promoter structure and cell cycle dependent expression of the human methylpurine-DNA glycosylase gene.

The methylpurine-DNA glycosylase (MPG) gene coding for human 3-methyladenine (3-meAde)-DNA glycosylase functions in the first step of base excision repair (BER) to remove numerous damaged bases including 3-meGua, ethenoadenine, and hypoxanthine (Hx) in addition to 3-meAde. In this report, we identify the length of the minimal MPG promoter region, demonstrate the involvement of several transcription factors in expression of the MPG gene, and determine the point at which transcription initiates. We also demonstrate that control of MPG expression is linked to MPG activity. To initiate studies on how the MPG functions with the ensemble of BER genes to effect repair, we have investigated the cell cycle control of MPG and other BER genes in normal human cells. Steady-state mRNA levels of MPG, human Nth homologue (NTH), and uracil-DNA glycosylase (UDG), DNA glycosylases, and human AP site-specific endonuclease (APE), an endonuclease incising DNA at abasic sites, are cell cycle dependent. In contrast, expression levels of genes coding for human 8-oxoguanine-DNA glycosylase (OGG1) and TDG DNA glycosylases, and omicron 6-methylguanine-DNA methyltransferase (MGMT) gene, and the RPA4 subunit gene do not vary with cell cycle. These observed cell cycle dependent differences might reflect distinct roles of individual BER proteins in mutation avoidance.

The DNA damage spectrum produced by simulated sunlight.

The mutagenic effects of ultraviolet and solar irradiation are thought to be due to the formation of DNA photoproducts, most notably cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts ((6-4)PPs). Experimental systems for determining the levels and sequence dependence of photoproduct formation in DNA have often used high doses of short-wave (UVC) irradiation. We have re-assessed this issue by using DNA sequencing technologies and different doses of UVC as well as more physiologically relevant doses of solar irradiation emitted from a solar UV simulator. It has been questioned whether hot alkali treatment can detect (6-4)PPs at all sequence positions. With high UVC doses, the sequence distribution of (6-4)PPs was virtually identical when hot alkali or UV damage endonuclease (UVDE) were used for detection, which appears to validate both methods. The (6-4)PPs form at 5'-TpC and 5'CpC sequences but very low levels are seen at all other dipyrimidines including 5'-TpT. Contrary to expectation, we find that (6-4) photoproducts form at almost undetectable levels under conditions of irradiation for up to five hours with the solar UV simulator. The same treatment produces high levels of CPDs. In addition, DNA glycosylases, which recognize oxidized and ring-opened bases, did not produce significant cleavage of sunlight-irradiated DNA. From these data, we conclude that cyclobutane pyrimidine dimers are at least 20 to 40 times more frequent than any other DNA photoproduct when DNA or cells are irradiated with simulated sunlight.

Mapping oxidative DNA damage and mechanisms of repair.

We developed a method to map oxidative-induced DNA damage at the nucleotide level using ligation-mediated polymerase chain reaction (LMPCR) technology. In vivo and in vitro DNA base modification patterns inflicted by reactive oxygen species (ROS) in the human P53 and PGK1 gene were nearly identical in vitro and in vivo. In human male fibroblasts, these patterns are independent of the transition metal used (Cu (II), Fe(II), or Cr(VI). Therefore, local probability of H2O2-mediated DNA base damage is determined primarily by DNA sequence. Moreover, in cells undergoing severe oxidative stress, extranuclear sites contribute metals that enhance nuclear DNA damage. The role of the base excision repair pathway in human cells responsible for the repair of the majority of ROS base damage is also discussed.

3-Methyladenine-DNA glycosylase (MPG protein) interacts with human RAD23 proteins.

Human 3-methyladenine-DNA glycosylase (MPG protein) initiates base excision repair by severing the glycosylic bond of numerous damaged bases. In comparison, homologues of the Rad23 proteins (hHR23) and the hXPC protein are involved in the recognition of damaged bases in global genome repair, a subset of nucleotide excision repair. In this report, we show that the hHR23A and -B also interact with the MPG protein and can serve as accessory proteins for DNA damage recognition in base excision repair. Furthermore, the MPG.hHR23 protein complex elevates the rate of MPG protein-catalyzed excision from hypoxanthine-containing substrates. This increased excision rate is correlated with a greater binding affinity of the MPG protein-hHR23 protein complex for damaged DNA. These data suggest that the hHR23 proteins function as universal DNA damage recognition accessory proteins in both of these major excision repair pathways.

Heterogeneous repair of N-methylpurines at the nucleotide level in normal human cells.

Base excision repair rates of dimethyl sulfate-induced 3-methyladenine and 7-methylguanine adducts were measured at nucleotide resolution along the PGK1 gene in normal human fibroblasts. Rates of 7-methylguanine repair showed a 30-fold dependence on nucleotide position, while position-dependent repair rates of 3-methyladenine varied only sixfold. Slow excision rates for 7-methylguanine bases afforded the opportunity to study their excision in vitro as a model for base excision repair. A two-component in vitro excision system, composed of human N-methylpurine-DNA glycosylase (MPG protein) and dimethyl sulfate-damaged DNA manifested sequence context-dependent rate differences for 7-methylguanine of up to 185-fold from position to position. This in vitro system reproduced both the global repair rate, and for the PGK1 coding region, the position-dependent repair patterns observed in cells. The equivalence of in vivo repair and in vitro excision data indicates that removal of 7-methylguanine by the MPG protein is the rate-limiting step in base excision repair of this lesion. DNA "repair rate footprints" associated with DNA glycosylase accessibility were observed only in a region with bound transcription factors. The "repair rate footprints" represent a rare chromatin component of 7-meG base excision repair otherwise dominated by sequence-context dependence. Comparison of in vivo repair rates to in vitro rates for 3-methyladenine, however, shows that the rate-limiting step determining position-dependent repair for this adduct is at one of the post-DNA glycosylase stages. In conclusion, this study demonstrates that a comparison of sequence context-dependent in vitro reaction rates to in vivo position-dependent repair rates permits the identification of steps responsible for position-dependent repair. Such analysis is now feasible for the different steps and adducts repaired via the base excision repair pathway.

Interaction of the recombinant human methylpurine-DNA glycosylase (MPG protein) with oligodeoxyribonucleotides containing either hypoxanthine or abasic sites.

Methylpurine-DNA glycosylases (MPG proteins, 3-methyladenine-DNA glycosylases) excise numerous damaged bases from DNA during the first step of base excision repair. The damaged bases removed by these proteins include those induced by both alkylating agents and/or oxidizing agents. The intrinsic kinetic parameters (k(cat) and K(m)) for the excision of hypoxanthine by the recombinant human MPG protein from a 39 bp oligodeoxyribonucleotide harboring a unique hypoxanthine were determined. Comparison with other reactions catalyzed by the human MPG protein suggests that the differences in specificity are primarily in product release and not binding. Analysis of MPG protein binding to the 39 bp oligodeoxyribonucleotide revealed that the apparent dissociation constant is of the same order of magnitude as the K(m) and that a 1:1 complex is formed. The MPG protein also forms a strong complex with the product of excision, an abasic site, as well as with a reduced abasic site. DNase I footprinting experiments with the MPG protein on an oligodeoxyribonucleotide with a unique hypoxanthine at a defined position indicate that the protein protects 11 bases on the strand with the hypoxanthine and 12 bases on the complementary strand. Competition experiments with different length, double-stranded, hypoxanthine-containing oligodeoxyribonucleotides show that the footprinted region is relatively small. Despite the small footprint, however, oligodeoxyribonucleotides comprising <15 bp with a hypoxanthine have a 10-fold reduced binding capacity compared with hypoxanthine-containing oligodeoxyribonucleotides >20 bp in length. These results provide a basis for other structural studies of the MPG protein with its targets.

Repair of DNA alkylation damage.

Alkylation damage of DNA is one of the major types of insults which cells must repair to remain viable. One way alkylation damaged ring nitrogens are repaired is via the Base Excision Repair (BER) pathway. Examination of mutants in both BER and Nucleotide Excision Repair show that there is actually an overlap of repair by these two pathways for the removal of cytotoxic lesions in Escherichia coli. The enzymes removing damaged bases in the first step in the BER pathway are DNA glycosylases. The coding sequences for a number of methylpurine-DNA glycosylases (MPG proteins) were cloned, and a comparison of the amino-acid sequences shows that there are some similarities between these proteins, but nonetheless, compared to other DNA glycosylases, MPG proteins are more divergent. MPG proteins have been purified to homogeneity and used to identify their substrates ranging from methylating agents to deamination products to oxidatively damaged bases. The ligation-mediated polymerase chain reaction has been used to study the formation of alkylation damage, and its repair in mammalian cells. We have studied DNA damage in the PGK1 gene for a series of DNA alkylating agents including N-methyl-N'-nitro-N-nitrosoguanidine, Mechlorethamine, and Chlorambucil and shown that the damage observed in the PGK1 (phosphoglycerate kinase 1) gene depends on the alkylating agent used. This report reviews the literature on the MPG proteins, DNA glycosylases removing 3-methyladenine, and the use of these enzymes to detect DNA damage at the nucleotide level.