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dc.contributor.authorSholder, Gabriel D.en_US
dc.date.accessioned2016-03-17T17:22:13Z
dc.date.available2016-03-17T17:22:13Z
dc.date.issued2014
dc.identifier.urihttps://hdl.handle.net/2144/15225
dc.description.abstractCells have lesion bypass DNA polymerases (DNAPs), often in the Y-Family, which synthesize passed DNA damage. One class of Y-Family DNAPs includes hDNAP k, EcDNAP IV and SsDbh, which insert accurately opposite N2-dG adducts, including BP-N2-dG formed from benzo[a]pyrene (BP). Another class includes hDNAP h, EcDNAP V and SsDpo4, which insert accurately opposite UV-damage. For correct Watson-Crick pairing between BP-N2-dG and dCTP, the BP moiety must be in the minor groove. On the minor groove side of the active site, k/IV/Dbh-class DNAPs have large openings that accommodate the BP moiety. Primer extension assays with purified proteins show that DNAP IV correctly inserts dCTP opposite BP more than 10-fold faster than it mis-inserts dATP, dGTP, or dTTP. In contrast, h/V/Dpo4-class DNAPs have small active site openings, which cannot accommodate BP and lead to a distorted structure and increased mutagenesis; e.g., Dpo4 has dGTP and dATP insertion rates that are 10-fold greater than those of dCTP. The opening in Dpo4 is plugged and bulky, whereas DNAP IV has a relatively spacious cavity. Consistent with this model, mutants of Dpo4 with a larger opening insert up to 10-fold more accurately opposite BP-N2-dG. Near the active site, Dpo4 has a single non-covalent bridge (NCB) between the little finger domain and the thumb-palm-fingers domain. DNAP IV and Dbh have a second, distal NCB that is 8 angstroms away from the active site towards the 3' end of the template DNA. Dpo4 becomes nearly 5-fold more accurate when mutated to carry a distal NCB, suggesting that NCB's also help control mutagenesis. Lastly, the active site of Dpo4 has a cavity in the major groove side, which may allow base flipping and dGTP insertion opposite -BP, while k/IV/Dbh-type polymerases do not. When this cavity is plugged in Dpo4 by mutagenesis or the introduction of an N-clasp motif, dGTP rates increase by nearly 20-fold. In conclusion, this data suggests that three structural regions contribute to accurate dCTP insertion opposite BP-N2-dG by k/IV/Dbh-class DNAPs: a large opening on the minor groove side near the active site, a cavity on the major groove side, and the number of non-covalent bridges between the little finger domain and the thumb-palm-fingers domain.en_US
dc.language.isoen_US
dc.subjectMolecular biologyen_US
dc.subjectBenzo[a]pyreneen_US
dc.subjectMutagenesisen_US
dc.subjectDNA polymeraseen_US
dc.subjectPrimer extension assayen_US
dc.subjectY-family polymeraseen_US
dc.titleY-family DNA polymerase architecture: three structural features control accurate deoxy CTP insertion opposite N2-deoxy-guanine-benzo-a-pyreneen_US
dc.typeThesis/Dissertationen_US
dc.date.updated2016-03-12T07:10:11Z
etd.degree.nameDoctor of Philosophyen_US
etd.degree.leveldoctoralen_US
etd.degree.disciplineMolecular Biology, Cell Biology & Biochemistryen_US
etd.degree.grantorBoston Universityen_US


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