Distinct catalytic activity and in vivo roles of the ExoIII and EndoIV AP endonucleases from Sulfolobus islandicus

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• 作者：Zhou Yan ; Qihong Huang ; Jinfeng Ni ; Yulong Shen
• 关键词：AP endonuclease ; Methyl methanesulfonate ; Base excision repair ; Archaea ; Sulfolobus islandicus
• 刊名：Extremophiles
• 出版年：2016
• 出版时间：September 2016
• 年：2016
• 卷：20
• 期：5
• 页码：785-793
• 全文大小：1,526 KB
• 刊物类别：Biomedical and Life Sciences
• 刊物主题：Life Sciences
  Microbiology
  Biotechnology
  Ecology
  
• 出版者：Springer Japan
• ISSN：1433-4909
• 卷排序：20

文摘

AP endonuclease cleaves the phosphodiester bond 5′- to the AP (apurinic or apyrimidinic) sites and is one of the major enzymes involved in base excision repair. So far, the properties of several archaeal AP endonuclease homologues have been characterized in vitro, but little is known about their functions in vivo. Herein, we report on the biochemical and genetic analysis of two AP endonucleases, SisExoIII and SisEndoIV, from the hyperthermophilic crenarchaeon Sulfolobus islandicus REY15A. Both SisExoIII and SisEndoIV exhibit AP endonuclease activity, but neither of them has 3′–5′ exonuclease activity. SisExoIII and SisEndoIV have similar K_M values on the substrate containing an AP site, but the latter cleaves the AP substrate at a dramatically higher catalytic rate than the former. Unlike other AP endonucleases identified in archaea, SisExoIII and SisEndoIV do not exhibit any cleavage activity on DNA having oxidative damage (8-oxo-dG) or uracil. Genetic analysis revealed that neither gene is essential for cell viability, and the growth of ∆SiRe_2666 (endoIV), ∆SiRe_0100 (exoIII), and ∆SiRe_0100∆SiRe_2666 is not affected under normal growth conditions. However, ∆SiRe_2666 exhibits higher sensitivity to the alkylating agent methyl methanesulfonate (MMS) than ∆SiRe_0100. Over-expression of SiRe_0100 can partially complement the sensitivity of ∆SiRe_2666 to MMS, suggesting a backup role of SisExoIII in AP site processing in vivo. Intriguingly, over-expression of SisEndoIV renders the strain more sensitive to MMS than the control. Taken together, we conclude that SisEndoIV, but not SisExoIII, is the main AP endonuclease that participates directly in base excision repair in S. islandicus.