Single domain shark VNAR antibodies neutralize SARS-CoV-2 infection in vitro.

Single domain shark variable domain of new antigen receptor (VNAR) antibodies can offer a viable alternative to conventional Ig-based monoclonal antibodies in treating COVID-19 disease during the current pandemic. Here we report the identification of neutralizing single domain VNAR antibodies selected against the severe acute respiratory syndrome coronavirus 2 spike protein derived from the Wuhan variant using phage display. We identified 56 unique binding clones that exhibited high affinity and specificity to the spike protein. Of those, 10 showed an ability to block both the spike protein receptor binding domain from the Wuhan variant and the N501Y mutant from interacting with recombinant angiotensin-converting enzyme 2 (ACE2) receptor in vitro. In addition, three antibody clones retained in vitro blocking activity when the E484K spike protein mutant was used. The inhibitory property of the VNAR antibodies was further confirmed for all 10 antibody clones using ACE2 expressing cells with spike protein from the Wuhan variant. The viral neutralizing potential of the VNAR clones was also confirmed for the 10 antibodies tested using live Wuhan variant virus in in vitro cell infectivity assays. Single domain VNAR antibodies, due to their low complexity, small size, unique epitope recognition, and formatting flexibility, should be a useful adjunct to existing antibody approaches to treat COVID-19.

The role of RuvA octamerization for RuvAB function in vitro and in vivo.

RuvA plays an essential role in branch migration of the Holliday junction by RuvAB as part of the RuvABC pathway for processing Holliday junctions in Escherichia coli. Two types of RuvA-Holliday junction complexes have been characterized: 1) complex I containing a single RuvA tetramer and 2) complex II in which the junction is sandwiched between two RuvA tetramers. The functional differences between the two forms are still not clear. To investigate the role of RuvA octamerization, we introduced three amino acid substitutions designed to disrupt the E. coli RuvA tetramer-tetramer interface as identified by structural studies. The mutant RuvA was tetrameric and interacted with both RuvB and junction DNA but, as predicted, formed complex I only at protein concentrations up to 500 nm. We present biochemical and surface plasmon resonance evidence for functional and physical interactions of the mutant RuvA with RuvB and RuvC on synthetic junctions. The mutant RuvA with RuvB showed DNA helicase activity and could support branch migration of synthetic four-way and three-way junctions. However, junction binding and the efficiency of branch migration of four-way junctions were affected. The activity of the RuvA mutant was consistent with a RuvAB complex driven by one RuvB hexamer only and lead us to propose that one RuvA tetramer can only support the activity of one RuvB hexamer. Significantly, the mutant failed to complement the UV sensitivity of E. coli DeltaruvA cells. These results indicate strongly that RuvA octamerization is essential for the full biological activity of RuvABC.

Hijacking of the human alkyl-N-purine-DNA glycosylase by 3,N4-ethenocytosine, a lipid peroxidation-induced DNA adduct.

Lipid peroxidation generates aldehydes, which react with DNA bases, forming genotoxic exocyclic etheno(epsilon)-adducts. E-bases have been implicated in vinyl chloride-induced carcinogenesis, and increased levels of these DNA lesions formed by endogenous processes are found in human degenerative disorders. E-adducts are repaired by the base excision repair pathway. Here, we report the efficient biological hijacking of the human alkyl-N-purine-DNA glycosylase (ANPG) by 3,N(4)-ethenocytosine (epsilonC) when present in DNA. Unlike the ethenopurines, ANPG does not excise, but binds to epsilonC when present in either double-stranded or single-stranded DNA. We developed a direct assay, based on the fluorescence quenching mechanism of molecular beacons, to measure a DNA glycosylase activity. Molecular beacons containing modified residues have been used to demonstrate that the epsilonC.ANPG interaction inhibits excision repair both in reconstituted systems and in cultured human cells. Furthermore, we show that the epsilonC.ANPG complex blocks primer extension by the Klenow fragment of DNA polymerase I. These results suggest that epsilonC could be more genotoxic than 1,N(6)-ethenoadenine (epsilonA) residues in vivo. The proposed model of ANPG-mediated genotoxicity of epsilonC provides a new insight in the molecular basis of lipid peroxidation-induced cell death and genome instability in cancer.

Characterisation of new substrate specificities of Escherichia coli and Saccharomyces cerevisiae AP endonucleases.

Despite the progress in understanding the base excision repair (BER) pathway it is still unclear why known mutants deficient in DNA glycosylases that remove oxidised bases are not sensitive to oxidising agents. One of the back-up repair pathways for oxidative DNA damage is the nucleotide incision repair (NIR) pathway initiated by two homologous AP endonucleases: the Nfo protein from Escherichia coli and Apn1 protein from Saccharomyces cerevisiae. These endonucleases nick oxidatively damaged DNA in a DNA glycosylase-independent manner, providing the correct ends for DNA synthesis coupled to repair of the remaining 5'-dangling nucleotide. NIR provides an advantage compared to DNA glycosylase-mediated BER, because AP sites, very toxic DNA glycosylase products, do not form. Here, for the first time, we have characterised the substrate specificity of the Apn1 protein towards 5,6-dihydropyrimidine, 5-hydroxy-2'-deoxyuridine and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine deoxynucleotide. Detailed kinetic comparisons of Nfo, Apn1 and various DNA glycosylases using different DNA substrates were made. The apparent K(m) and kcat/K(m) values of the reactions suggest that in vitro DNA glycosylase/AP lyase is somewhat more efficient than the AP endonuclease. However, in vivo, using cell-free extracts from paraquat-induced E.coli and from S.cerevisiae, we show that NIR is one of the major pathways for repair of oxidative DNA base damage.

1,N(2)-ethenoguanine, a mutagenic DNA adduct, is a primary substrate of Escherichia coli mismatch-specific uracil-DNA glycosylase and human alkylpurine-DNA-N-glycosylase.

The promutagenic and genotoxic exocyclic DNA adduct 1,N(2)-ethenoguanine (1,N(2)-epsilonG) is a major product formed in DNA exposed to lipid peroxidation-derived aldehydes in vitro. Here, we report that two structurally unrelated proteins, the Escherichia coli mismatch-specific uracil-DNA glycosylase (MUG) and the human alkylpurine-DNA-N-glycosylase (ANPG), can release 1,N(2)-epsilonG from defined oligonucleotides containing a single modified base. A comparison of the kinetic constants of the reaction indicates that the MUG protein removes the 1,N(2)-epsilonG lesion more efficiently (k(cat)/K(m) = 0.95 x 10(-3) min(-1) nm(-1)) than the ANPG protein (k(cat)/K(m) = 0.1 x 10(-3) min(-1) nm(-1)). Additionally, while the nonconserved, N-terminal 73 amino acids of the ANPG protein are not required for activity on 1,N(6)-ethenoadenine, hypoxanthine, or N-methylpurines, we show that they are essential for 1,N(2)-epsilonG-DNA glycosylase activity. Both the MUG and ANPG proteins preferentially excise 1,N(2)-epsilonG when it is opposite dC; however, unlike MUG, ANPG is unable to excise 1,N(2)-epsilonG when it is opposite dG. Using cell-free extracts from genetically modified E. coli and murine embryonic fibroblasts lacking MUG and mANPG activity, respectively, we show that the incision of the 1,N(2)-epsilonG-containing duplex oligonucleotide has an absolute requirement for MUG or ANPG. Taken together these observations suggest a possible role for these proteins in counteracting the genotoxic effects of 1,N(2)-epsilonG residues in vivo.

Repair of oxidized purines and damaged pyrimidines by E. coli Fpg protein: different roles of proline 2 and lysine 57 residues.

The Escherichia coli Fpg protein is involved in the repair of oxidized purines, including the highly mutagenic 7,8-dihydro-8-oxoguanine (8-oxoG). The Fpg protein also excises various oxidized pyrimidines with high efficiency. We examined, by targeted mutagenesis, the role of two highly conserved amino acid residues, proline 2 (P2) and lysine 57 (K57), on the catalytic activities of the Fpg protein toward a ring-fragmentation product of thymine (alpha RT) and 5,6-dihydrothymine (dHT). The following E. coli mutant Fpg proteins were investigated: lysine 57 --> glycine (FpgK57G), proline 2 --> glycine (FpgP2G), and proline 2 --> glutamic acid (FpgP2E). The FpgK57G protein had barely detectable alpha RT and dHT-DNA glycosylase activities and produced minute amounts of a Schiff-base complex upon reaction with alpha RT containing DNA. In contrast, the activity of an FpgP2G mutant toward alpha RT was comparable to the wild type activity and produced a Schiff-base complex with this substrate. FpgP2E was completely inactive in all the assays, in contrast, to the other mutants. The crystal structure of a homologous Fpg protein from an extreme thermophile, Thermus thermophilus HB8, reveals that it is composed of two distinct domains connected by a flexible hinge (Sugahara et al. [2000]: EMBO J 19:3857-3869). The N-terminal proline, one primary residue for enzymatic catalysis, is positioned at the bottom of a cleft in close proximity to lysine 52 (analogous to K57 of the E. coli Fpg). Based on the biochemical assays, together with the crystal structure of T. thermophilus HB8 Fpg protein, we propose a two-nucleophile model for the enzymatic catalysis.