Specificity of damage-bypass DNA polymerases E. coli DinB and human pol κ
Permanent URL:
http://hdl.handle.net/2047/D20260514
Ondrechen, Mary Jo (Committee member)
Mattos, Carla (Committee member)
Lone, Samer (Committee member)
Using site-directed mutagenesis we have created alanine mutations in the active site loop 1, adjacent to the incoming nucleotide, of both DinB and pol κ. These mutations resulted in a range of activity and fidelity opposite undamaged DNA and the preferred lesion of both polymerases, N2. The variants that retained activity similar to the wild-type proteins had decreased fidelity, especially opposite N2-furfuryl-dG while the variants with decreased activity did not show a decrease in fidelity.
Both E. coli DinB and human pol κ are able to bypass minor groove adducts on the N2 position of deoxyguanine efficiently and correctly while adducts in the major groove, such as the N6 position of deoxyadenine are blocking lesions. However, another DinB ortholog, archaeal Dpo4 can bypass major groove adducts. The active site loop of these three proteins differs in length and in order to determine if the active site loops are important for damage specificity we created loop swap chimeras of the three proteins. We found that pol κ is tolerant to increases in length of its active site loop but DinB is not tolerant to increases or decreases in the length of its active site loop.
Y-family DNA polymerases have been implicated in antibiotic resistance as well as cancer and chemotherapy resistance. We found that two pol κ mutations found in cancer tumors have an increase in insertion and extension activity as well as decreases in fidelity opposite both minor groove and major groove adducts. It is believed that Y-family DNA polymerases could be targets to increase chemotherapeutic efficacy and decrease antibiotic resistance. Using a computational molecular modeling screen, a number of potential inhibitors of DinB were identified and we found that the compounds inhibit DinB at much lower concentrations than pol κ, and many do not inhibit pol κ even at high concentrations.
The work presented here reveals the important roles of the active site loop in DinB and pol in activity and fidelity. Point mutations in the DinB active site loop have a range of effects, whereas changing the length of the loop results in dramatic losses of activity. In contrast, human pol is far more tolerant to changes in its active site loop as well to potential inhibitors. This work suggests that DinB is overall more stringent in its lesion bypass activities than pol κ.
E. coli
pol κ
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