DNA polymerase beta (pol beta) is the main polymerase involved in base excision repair (BER), which is a pathway responsible for the repair of tens of thousands of DNA lesions per cell per day. Our recent efforts in sequencing colon tumors showed that 40% of the tumors sequenced possessed a variant in the coding region of the POLB gene, one of these variants is E288K. Expression of the E288K variant in cells leads to an increase in mutations at AT base pairs. In vitro, the E288K variant is as active as and binds one-base gapped DNA with the same affinity as wild-type pol beta. Single turnover kinetic data on the E288K variant show that its mutator phenotype is specific for misincorporating opposite template A up to 6-fold more than the wild-type enzyme and that this is due to a decrease in discrimination in nucleotide binding. Molecular modeling suggests that the substituation of Lys at position 288 causes the polymerase to adopt a more open conformation, which may be disrupting the nucleotide binding pocket. This may explain the reduced discrimination at the level of nucleotide binding. The increased mutagenesis of the E288K variant could lead to genomic instability and ultimately a malignant tumor phenotype.