Role of the core in CTnDOT integration

Tyrosine recombinase;CTnDOT;Integration;Conjugative transposon

Laprise, Jennifer

关键词: Tyrosine recombinase;    CTnDOT;    Integration;    Conjugative transposon;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/34474/LAPRISE_JENNIFER.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

IntDOT is the tyrosine recombinase encoded by the Bacteroides conjugative transposon CTnDOT. It catalyzes integration and excision into and out of the bacterial host chromosome. Although it is a member of the well studied tyrosine recombinase family, IntDOT is of particular interest because it can catalyze recombination between substrates containing heterology in the overlap region. This tolerance for heterology suggests that IntDOT uses a different mechanism for recombination. IntDOT performs initial cleavage on the top strand adjacent to the D and B core sites. The first two bases within the overlap region are a conserved GC dinucleotide that provide the only sequence identity between the two substrates. I used complementary DNA oligonucleotides to invert the overlap region 180 degrees to relocate the GC dinucleotide to the bottom strand adjacent to the B’ core site of attB. I tested the inverted overlap attB site in an in vitro integration assay with wild-type attDOT and showed that integration still occurs, albeit in the opposite orientation relative to the wild type reaction. I used nicked attB substrates to show that initial cleavage of the inverted overlap attB substrate takes place on the bottom strand adjacent to the B’ core site, suggesting that the location of sequence identity within the overlap determines the site of initial cleavage. I confirmed this hypothesis by using an attB site containing a symmetric overlap sequence.