Excision of 5-hydroxymethyluracil in single-stranded DNA by hSMUG1 requires Ser26 and/or Glu35 and Pro240
Master thesis
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https://hdl.handle.net/11250/2780576Utgivelsesdato
2021Metadata
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Sammendrag
The integrity of the genetic material of all organisms is assaulted continuously by a multitude of endogenous and exogenous agents throughout its lifetime, where the DNA bases of the genetic code may be altered if not repaired properly before replication causing cellular malfunction, cell death or cancer. Uracil (U), which is a normal base in RNA, arises in DNA by hydrolytic deamination of cytosine (C) or by erroneous incorporation of deoxyuridine monophosphate opposite adenine (A) during replication. Other damaging events is caused by reactive oxygen species (ROS) generated during aerobic metabolism, as e.g., oxidation of thymine (T) into 5-hydroxymethyluracil (hmU), which may disrupt normal gene expression if left unrepaired. Cells have developed a multitude of repair mechanisms to correct each type of DNA damage, where uracil-DNA glycosylases (UDGs) repair U and U-derivatives by initiating the base excision repair (BER) pathway. One UDG is single-strand selective monofunctional uracil-DNA glycosylase (SMUG1), which excises U and hmU from DNA in addition to several other bases because of its flexible active site. In the study we found that replacing certain residues (Ser26/Glu35 and Pro240) in human SMUG1 eliminated the excision activity for hmU in single-stranded (ss) DNA, while the excision activity for uracil was retained in ssDNA, bubble DNA and R-loop DNA as demonstrated using commercial as well as our own purified enzyme. This is the first study showing the importance of these amino acid residues for the excision of hmU from DNA by SMUG1.