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Date of Award

Winter 1-15-2021

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Biochemistry)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

E. coli RecBCD is crucial in initiating repair of double stranded (ds) DNA breaks. It is a heterotrimeric helicase and nuclease complex possessing two ATPase motors, RecB and RecD, and a regulatory subunit without ATPase activity, RecC. The RecB subunit also contains a 30kDa nuclease domain (RecBNuc) that, according to published structural data, is situated over 60Å away from the site of dsDNA binding. Surprisingly, we have shown in previous studies that deletion of RecBNuc to form RecBΔNucCD affects its dsDNA unwinding properties. The mechanism by which RecBNuc influences RecBCD dsDNA unwinding is unclear. In this thesis, equilibrium binding techniques, specifically fluorescence titration and isothermal titration calorimetry experiments, were used to examine the thermodynamics of RecBCD binding to DNA ends and particularly how it is affected by removal of RecBNuc as a function of Na+, Mg2+ and DNA ends possessing various lengths of dT tails (on either 3’ or 5’ end, or both). Cryo-EM was also employed to examine any potential conformational changes upon RecBCD or RecBΔNucCD binding to blunt-ended DNA. While equilibrium binding affinities determined in this work agree with previous studies showing that RecBCD binds optimally to duplex DNA possessing ssDNA tails with lengths of 3’-dT6 and 5’-dT10, I also found that the favorable ΔH for binding requires longer ssDNA tails (3’-dT10 and 5’-dT15) to reach a plateau. This indicates that RecBCD can form favorable interactions with ssDNA tails that are longer than previously thought. These additional interactions are obscured by an enthalpy-entropy compensation. Furthermore, the observed ΔH for RecBCD binding to dsDNA ends possessing 3’-dTL and 5’-dTL tails of the same length (twin-tailed substrates) reaches a plateau at an even longer tail length of L=17-18. These favorable interactions can also represent favorable enthalpic components compensating for the enthalpic cost of DNA melting. Thus my results suggests that RecBCD can melt 9-11bp at a blunt DNA end and even up to 17-18bp. My results also indicate that the energetic contributions of the 3’ and 5’ tails to RecBCD binding are not independent and that additional interactions with longer ssDNA tails occur when both 3’ and 5’ tails interact with RecBCD. Interestingly, my studies with RecBΔNucCD indicate that it binds with higher affinity than RecBCD to all DNA ends by up to 15-fold. The removal of the RecBNuc domain also results in values of ΔH that reach a plateau at dT15 for twin-tailed DNA substrates, indicating that the RecBNuc influences the interactions of RecBCD with a DNA end possessing twin ssDNA tails longer than dT15. Surprisingly, my Cryo-EM structures indicate that RecBCD can melt at least 11 bp upon binding to a blunt-ended DNA duplex in the presence of Mg2+, whereas previous crystal structures showed RecBCD melting no more than 4-6 bp upon binding to blunt-ended DNA. In the absence of RecBNuc, the 1A and 2A sub-domains of RecD become much more flexible and show weak density under Cryo-EM. Because of such conformation flexibility, only 3 bp of DNA melting can be observed for RecBΔNucCD. In addition, density of RecBNuc is entirely missing among 55% of RecBCD (DNA-unbound) particles. Given that both sedimentation velocity and denaturing gels show intact RecBCD complexes, this indicates that RecBNuc is often undocked from the location that is seen in other published structures. However, I did not observe RecBNuc docking at any alternative sites. I found that DNA binding significantly enhances the density of RecD and RecBNuc. Interestingly, RecD shows weak density in all particles of RecBΔNucCD both with and without DNA. These results indicate that both RecBNuc and DNA binding are important for stabilizing conformations of RecD while DNA binding also helps keeping RecBNuc docked on RecC. This thesis presents data that argues an important role of RecBNuc in allosteric communication between RecB and RecD subunits and in stabilizing the conformations of RecD subunit in both DNA bound and unbound states. This helps us understand how removal of RecBNuc influences the initiation of RecBCD dsDNA unwinding.

Language

English (en)

Chair and Committee

Timothy Lohman

Committee Members

Peter Burgers, Roberto Galletto, Eric Galburt, Nima Mosammaparast,

Available for download on Friday, December 31, 2021

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