Contribution of distal residues in Escherichia coli DinB and human DNA polymerase κ to their activities in translesion synthesis

• Online Access: http://hdl.handle.net/2047/D20413927

DNA is constantly threatened by 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 lesions when copying damaged DNA in a process called translesion synthesis. DinB is one of these polymerases, which is involved in bypassing deoxyguanosine adducts at the N2 position. There are also four human Y family polymerases, including DNA polymerase kappa, that have similar function. E. coli DinB and human pol kappa both bypass minor groove adducts and are inhibited by major groove adducts. However, pol κ is more efficient in the extension step of translesion synthesis (TLS). In order to investigate the importance of particular residues in the extension step of TLS, the computational method POOL was utilized. This tool identified not only active site residues and residues previously observed to be important for activity, but it also predicted more distant residues that do not have direct contact with substrate that may have catalytic importance. These residues are in different regions of DinB and pol κ, which could provide insight into the differences in their activities. To study the contribution of these distal residues on the extension step of TLS, DinB and pol κ variants with mutations at the predicted distal positions were constructed and have been assayed for bypass of damage lesions. There is also a loop in pol κ that is unresolved in all published crystal structures. This loop has been deleted from pol κ to understand its impact on pol κ stability and activity. The variants containing mutations at POOL-predicted residues in DinB showed compromised extension activity of not only DinB but also at three positions in pol κ. The majority of variants harboring mutations at POOL-predicted residues in pol κ did not affect activity of either pol κ or DinB. Pol κ with the unresolved loop deleted also did not have altered activity or stability compared with wild-type pol κ, which has further implications for computational studies.--Author's abstract