This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. In 2010, we have collected a number of X-ray diffraction data sets from crystals of ternary complexes (TCs) formed by the wild type and mutant bacteriophage RB69 DNA polymerase (RB69pol) at NE-CAT, APS, resulting in two publications (Wang et al., Insights into base selectivity from the 1.8 ? resolution structure of an RB69 DNA polymerase ternary complex, Biochemistry. in press;Xia et al., Variation in mutation rates caused by RB69pol fidelity mutants can be rationalized on the basis of their kinetic behavior and crystal structures. J. Mol. Biol. In press). In the first publication, we have extended the resolution of RB69pol TCs from our previously published 2.6 ?-resolution structure to the currently 1.8 ?-resolution structure, and revealed important functional roles of ordered-water molecules mediating the polymerase-DNA duplex interactions in incorporation efficiency of dNMPs. Using a strategically positioned mispair in DNA duplexes, we showed that primer-extension decreased by 100 fold after moving the mispair from the fourth to the third basepair, and sequentially by another 10 fold to the second and first basepair of the primer-terminus and finally to the nascent base pair position. In the second publication, we have captured the replication-competent TCs formed by an RB69pol mutant containing the dG/dTTP mispair at the polymerization insertion site. This is the first structure of the replication-competent TCs formed by any polymerases containing incorrect incoming dNTPs at the polymerization insertion site. This allows us to correlate crystal structures of a replicative polymerase to the kinetic behavior for incorporation of incorrect dNMPs as well as to correlate in vivo mutational rates with relevant in vitro kinetic parameters.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR015301-09
Application #
8361718
Study Section
Special Emphasis Panel (ZRG1-BCMB-K (40))
Project Start
2011-04-01
Project End
2012-03-31
Budget Start
2011-04-01
Budget End
2012-03-31
Support Year
9
Fiscal Year
2011
Total Cost
$3,666
Indirect Cost
Name
Cornell University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
872612445
City
Ithaca
State
NY
Country
United States
Zip Code
14850
Chen, Wenyang; Mandali, Sridhar; Hancock, Stephen P et al. (2018) Multiple serine transposase dimers assemble the transposon-end synaptic complex during IS607-family transposition. Elife 7:
Eichhorn, Catherine D; Yang, Yuan; Repeta, Lucas et al. (2018) Structural basis for recognition of human 7SK long noncoding RNA by the La-related protein Larp7. Proc Natl Acad Sci U S A 115:E6457-E6466
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Krotee, Pascal; Rodriguez, Jose A; Sawaya, Michael R et al. (2017) Atomic structures of fibrillar segments of hIAPP suggest tightly mated ?-sheets are important for cytotoxicity. Elife 6:
Dhayalan, Balamurugan; Mandal, Kalyaneswar; Rege, Nischay et al. (2017) Scope and Limitations of Fmoc Chemistry SPPS-Based Approaches to the Total Synthesis of Insulin Lispro via Ester Insulin. Chemistry 23:1709-1716
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Kattke, Michele D; Chan, Albert H; Duong, Andrew et al. (2016) Crystal Structure of the Streptomyces coelicolor Sortase E1 Transpeptidase Provides Insight into the Binding Mode of the Novel Class E Sorting Signal. PLoS One 11:e0167763
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