Structural and Functional Studies of DNA Nucleases: SgrAI and Mk0566

Persistent Link:
http://hdl.handle.net/10150/311237
Title:
Structural and Functional Studies of DNA Nucleases: SgrAI and Mk0566
Author:
Shah, Santosh
Issue Date:
2013
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Embargo:
Release 25-Dec-2014
Abstract:
DNA nucleases are essential for various biological functions such as replication, recombination, and repair. Restriction endonucleases (REs) are excellent model system for the investigation of DNA recognition and specificity. SgrAI is a type IIF RE that cuts an 8 base pair primary sequence. In addition to its primary cleavage activity it also cleaves secondary sequences, but only appreciably in the presence of the primary sequence. The longer flanking DNA exhibits much greater activated DNA cleavage by SgrAI (>1000 fold activation by secondary site). Interestingly, the asymmetric cleavage seen in one of the two types of secondary site DNA is lost upon activation of SgrAI, suggesting a loss of communication between DNA recognition and activity upon specificity expansion. The structure of SgrAI bound to 22-1HT supports the cryoelectron microscopy structure of activated, oligomeric SgrAI highlighting the significance of the contacts made by the flanking DNA and the role played by N-terminal domain contacts in forming the run-on oligomer. The biological study suggests that the run-on oligomer formation sequesters the host DNA from being cleaved by the activated SgrAI complex. The DNA sequence binding, cleavage preference, and the structure of K96A SgrAI were determined. Unexpectedly, this mutation did not alter the structure of the enzyme, nor did it result in an enzyme lacking sequence preference at the 7ᵗʰ position. Instead, the largest effect of the mutation appears to be in making the enzyme more specific such that it fails to cleave either type of secondary site. It may be that the K96 side chain is required to distort the non YG sequences (specifically GG and TC) of secondary site DNA for proper positioning in the enzyme active site upon activation and specificity expansion. The crystal structure of Mk0566, XPG homologue from M. kandleri, was solved to 2.48 Å resolution and was found to be very similar to that of human FEN-1 and to other archaeal FEN-1/XPG homologues. These results suggest that the main biological role of Mk0566 is in DNA replication; however, they do not preclude involvement in a modified form of nucleotide excision repair.
Type:
text; Electronic Dissertation
Keywords:
Allostery; Indirect readout mechanism; Nucleotide excision repair; Restriction endonuclease; Run-on oligomerization; Biochemistry; Activation
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Graduate College; Biochemistry
Degree Grantor:
University of Arizona
Advisor:
Horton, Nancy C.

Full metadata record

DC FieldValue Language
dc.language.isoen_USen_US
dc.titleStructural and Functional Studies of DNA Nucleases: SgrAI and Mk0566en_US
dc.creatorShah, Santoshen_US
dc.contributor.authorShah, Santoshen_US
dc.date.issued2013-
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.releaseRelease 25-Dec-2014en_US
dc.description.abstractDNA nucleases are essential for various biological functions such as replication, recombination, and repair. Restriction endonucleases (REs) are excellent model system for the investigation of DNA recognition and specificity. SgrAI is a type IIF RE that cuts an 8 base pair primary sequence. In addition to its primary cleavage activity it also cleaves secondary sequences, but only appreciably in the presence of the primary sequence. The longer flanking DNA exhibits much greater activated DNA cleavage by SgrAI (>1000 fold activation by secondary site). Interestingly, the asymmetric cleavage seen in one of the two types of secondary site DNA is lost upon activation of SgrAI, suggesting a loss of communication between DNA recognition and activity upon specificity expansion. The structure of SgrAI bound to 22-1HT supports the cryoelectron microscopy structure of activated, oligomeric SgrAI highlighting the significance of the contacts made by the flanking DNA and the role played by N-terminal domain contacts in forming the run-on oligomer. The biological study suggests that the run-on oligomer formation sequesters the host DNA from being cleaved by the activated SgrAI complex. The DNA sequence binding, cleavage preference, and the structure of K96A SgrAI were determined. Unexpectedly, this mutation did not alter the structure of the enzyme, nor did it result in an enzyme lacking sequence preference at the 7ᵗʰ position. Instead, the largest effect of the mutation appears to be in making the enzyme more specific such that it fails to cleave either type of secondary site. It may be that the K96 side chain is required to distort the non YG sequences (specifically GG and TC) of secondary site DNA for proper positioning in the enzyme active site upon activation and specificity expansion. The crystal structure of Mk0566, XPG homologue from M. kandleri, was solved to 2.48 Å resolution and was found to be very similar to that of human FEN-1 and to other archaeal FEN-1/XPG homologues. These results suggest that the main biological role of Mk0566 is in DNA replication; however, they do not preclude involvement in a modified form of nucleotide excision repair.en_US
dc.typetexten_US
dc.typeElectronic Dissertationen_US
dc.subjectAllosteryen_US
dc.subjectIndirect readout mechanismen_US
dc.subjectNucleotide excision repairen_US
dc.subjectRestriction endonucleaseen_US
dc.subjectRun-on oligomerizationen_US
dc.subjectBiochemistryen_US
dc.subjectActivationen_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.disciplineBiochemistryen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorHorton, Nancy C.en_US
dc.contributor.committeememberHorton, Nancy C.en_US
dc.contributor.committeememberMontfort, William R.en_US
dc.contributor.committeememberCordes, Matthew H. J.en_US
dc.contributor.committeememberMcEvoy, Megan M.en_US
dc.contributor.committeememberGhosh, Indraneelen_US
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