RESUMO
DNA interstrand crosslinks (ICLs) strands pose an impenetrable barrier for DNA replication. Different ICLs are known to recruit distinct DNA repair pathways. NEIL3 glycosylase has been known to remove an abasic (Ap) site derived DNA crosslink (Ap-ICL). An Ap-ICL forms spontaneously from the Ap site with an adjacent adenine in the opposite strand. Lack of genetic models and a poor understanding of the fate of these lesions leads to many questions about the occurrence and the toxicity of Ap-ICL in cells. Here, we investigate the circumstances of Ap-ICL formation. With an array of different oligos, we have investigated the rates of formation, the yields, and the stability of Ap-ICL. Our findings point out how different bases in the vicinity of the Ap site change crosslink formation in vitro. We reveal that AT-rich rather than GC-rich regions in the surrounding Ap site lead to higher rates of Ap-ICL formation. Overall, our data reveal that Ap-ICL can be formed in virtually any DNA sequence context surrounding a hot spot of a 5'-Ap-dT pair, albeit with significantly different rates and yields. Based on Ap-ICL formation in vitro, we attempt to predict the number of Ap-ICLs in the cell.
Assuntos
Replicação do DNA , DNA , Reagentes de Ligações Cruzadas/toxicidade , Dano ao DNA , Reparo do DNARESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease-19 pandemic. One of the key components of the coronavirus replication complex are the RNA methyltransferases (MTases), RNA-modifying enzymes crucial for RNA cap formation. Recently, the structure of the 2'-O MTase has become available; however, its biological characterization within the infected cells remains largely elusive. Here, we report a novel monoclonal antibody directed against the SARS-CoV-2 non-structural protein nsp10, a subunit of both the 2'-O RNA and N7 MTase protein complexes. Using this antibody, we investigated the subcellular localization of the SARS-CoV-2 MTases in cells infected with the SARS-CoV-2.
Assuntos
COVID-19/virologia , Metiltransferases/metabolismo , Capuzes de RNA/genética , RNA Viral/genética , SARS-CoV-2/enzimologia , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Anticorpos Monoclonais/análise , Humanos , Metiltransferases/análise , Metiltransferases/genética , Transporte Proteico , Capuzes de RNA/metabolismo , RNA Viral/metabolismo , SARS-CoV-2/química , SARS-CoV-2/genética , Proteínas não Estruturais Virais/análise , Proteínas não Estruturais Virais/genética , Proteínas Virais Reguladoras e Acessórias/análise , Proteínas Virais Reguladoras e Acessórias/genéticaRESUMO
Bacterial MutM is a DNA repair glycosylase removing DNA damage generated from oxidative stress and, therefore, preventing mutations and genomic instability. MutM belongs to the Fpg/Nei family of prokaryotic enzymes sharing structural and functional similarities with their eukaryotic counterparts, for example, NEIL1-NEIL3. Here, we present two crystal structures of MutM from pathogenic Neisseria meningitidis: a MutM holoenzyme and MutM bound to DNA. The free enzyme exists in an open conformation, while upon binding to DNA, both the enzyme and DNA undergo substantial structural changes and domain rearrangement. Our data show that not only NEI glycosylases but also the MutMs undergo dramatic conformational changes. Moreover, crystallographic data support the previously published observations that MutM enzymes are rather flexible and dynamic molecules.
