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Biochemical and structural characterization of a Type IV restriction endonuclease SauUSI

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dc.contributor.advisor KAYARAT, SAIKRISHNAN en_US
dc.contributor.author TUMULURI, VINAYAK SADASIVAM en_US
dc.date.accessioned 2022-10-11T10:08:03Z
dc.date.available 2022-10-11T10:08:03Z
dc.date.issued 2022-08 en_US
dc.identifier.citation 142 en_US
dc.identifier.uri http://dr.iiserpune.ac.in:8080/xmlui/handle/123456789/7394
dc.description.abstract Restriction modification enzymes are major barriers to horizontal gene transfer (HGT) in bacteria. These enzymes are diverse in the nature of targets that they recognize, their co-factor requirements and mechanisms of action. They are broadly classified as modification independent and modification dependent restriction enzymes. SauUSI is a modification dependent restriction endonuclease and is known to thwart the acquisition of Vancomycin resistance genes in Staphylococcus aureus. Here, I report a detailed biochemical and structural characterization of SauUSI where in the presence of ATP the enzyme can cleave DNA having a single or multiple target site/s. Remarkably in substrates with two or more target sites, the DNA region flanked by two target sites is shred into smaller fragments. I also report the first crystal structure of SauUSI which reveals a stable dimer with the interface at the nuclease domains of the two protomers. The architecture of SauUSI facilitates cleavage of both DNA with a single or multiple target site/s and provides a molecular basis for target recognition. ATP hydrolysis powers DNA translocation and we propose that in a DNA flanked by two target sites, translocating SauUSI molecules pile up against a target site bound SauUSI molecule eventually leading to multiple double stranded DNA breaks causing irreparable DNA shredding. The lesser efficient single-site cleavage is attributed to a switch-based mechanism. I also report for the first time that an ATP dependent restriction endonuclease; SauUSI, functions as a bona fide single-stranded DNA nuclease. Interestingly, this nucleolytic activity is target-site and nucleotide independent. Further, SauUSI functions as an endonuclease on ssDNA because of its ability to cleave substrates with gaps and mismatch bubbles. However, this nuclease activity is restricted to ssDNA as ssRNA is refractory to cleavage by SauUSI. This ssDNA endonucleolytic activity of SauUSI is downregulated by the presence of single-stranded DNA binding protein and Adenine nucleotides such as ATP, dATP and ADP, thus providing a paradigm for protecting bacterial self-DNA. I hypothesize that this ssDNA cleavage activity could act as a barrier to modes of HGT such as transduction involving ssDNA phages, conjugation and natural transformation. en_US
dc.language.iso en en_US
dc.subject Biochemistry en_US
dc.subject Structural Biology en_US
dc.title Biochemical and structural characterization of a Type IV restriction endonuclease SauUSI en_US
dc.type Thesis en_US
dc.description.embargo one year en_US
dc.type.degree Int.Ph.D en_US
dc.contributor.department Dept. of Biology en_US
dc.contributor.registration 20152019 en_US


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  • PhD THESES [602]
    Thesis submitted to IISER Pune in partial fulfilment of the requirements for the degree of Doctor of Philosophy

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