ABSTRACT
The recent outbreak of COVID-19 has created a serious health crisis with fatFal infectious viral diseases, such as Severe Acute Respiratory Syndrome (SARS). The nsp13, a helicase of coronaviruses is an essential element for viral replication that unwinds secondary structures of DNA and RNA, and is thus considered a major therapeutic target for treatment. The replication of coronaviruses and other retroviruses occurs in the cytoplasm of infected cells, in association with viral replication organelles, called virus-induced cytosolic double-membrane vesicles (DMVs). In addition, an increase in cytosolic Ca2+ concentration accelerates viral replication. However, the molecular mechanism of nsp13 in the presence of Ca2+ is not well understood. In this study, we applied biochemical methods and single-molecule techniques to demonstrate how nsp13 achieves its unwinding activity while performing ATP hydrolysis in the presence of Ca2+. Our study found that nsp13 could efficiently unwind double stranded (ds) DNA under physiological concentration of Ca2+ of cytosolic DMVs. These findings provide new insights into the properties of nsp13 in the range of calcium in cytosolic DMVs.
Subject(s)
Calcium , DNA , Nucleic Acid Conformation , RNA Helicases , Single Molecule Imaging , Viral Nonstructural Proteins , Calcium/metabolism , Calcium/pharmacology , DNA/chemistry , DNA/drug effects , DNA/metabolism , Magnesium/metabolism , Magnesium/pharmacology , Nucleic Acid Conformation/drug effects , Adenosine Triphosphate/metabolism , Virus Replication , Cytosol/metabolism , Hydrolysis/drug effects , RNA Helicases/drug effects , RNA Helicases/metabolism , Viral Nonstructural Proteins/drug effects , Viral Nonstructural Proteins/metabolism , Fluorescence Resonance Energy Transfer , Electrophoresis, Polyacrylamide Gel , Dose-Response Relationship, Drug , Transcription, GeneticABSTRACT
During base excision repair, a transient single-stranded DNA (ssDNA) gap is produced at the apurinic/apyrimidinic (AP) site. Exonuclease III, capable of performing both AP endonuclease and exonuclease activity, are responsible for gap creation in bacteria. We used single-molecule fluorescence resonance energy transfer to examine the mechanism of gap creation. We found an AP site anchor-based mechanism by which the intrinsically distributive enzyme binds strongly to the AP site and becomes a processive enzyme, rapidly creating a gap and an associated transient ssDNA loop. The gap size is determined by the rigidity of the ssDNA loop and the duplex stability of the DNA and is limited to a few nucleotides to maintain genomic stability. When the 3' end is released from the AP endonuclease, polymerase I quickly initiates DNA synthesis and fills the gap. Our work provides previously unidentified insights into how a signal of DNA damage changes the enzymatic functions.
