Probing the role of distal residues in DinB and Pol Kappa in the extension step of DNA damage bypass

Abstract

DNA is constantly subjected to damage from endogenous and exogenous sources. Replicative DNA polymerases are typically unable to replicate damaged DNA, but specialized DNA polymerases in the Y family possess this ability. Escherichia coli has two Y family polymerases that are specialized to bypass damage when copying DNA in a process called translesion synthesis (TLS). DinB is one of these polymerases and is involved in bypassing deoxyguanosine adducts at the N2 position. Humans have four Y family polymerases, including DNA polymerase kappa. E. coli DinB and human pol kappa both bypass minor groove adducts and are inhibited by major groove adducts. However, pol kappa is more efficient in copying past DNA damage in the extension step of translesion synthesis. In order to probe the importance of particular residues in the extension step of TLS, the computational tool POOL was utilized. This method identified active site residues and residues previously observed to be important for activity. POOL also predicted more distant residues that do not have direct contact with substrates that may have catalytic importance, but the residues are in different regions of DinB and pol kappa. To study the contribution of these distal residues on the extension step of TLS, DinB and pol kappa variants with mutations at the predicted distal positions were constructed and are being assayed for bypass of damage. We have identified variants with a range of activity on undamaged and damaged DNA; in particular several mutations in the DinB little finger domain severely reduce activity.Support or Funding InformationSupport from NSF‐MCB‐1517290, American Cancer Society RSG‐12‐161‐01‐DMC, and the PhRMA Foundation (predoctoral fellowship in informatics awarded to CLM)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.