DNA polymerase ␤ (pol ␤) is a 39-kDa protein that functions in DNA repair processes in mammalian cells. As a first step toward understanding mechanisms of polymerase fidelity, we developed a genetic method to identify mammalian pol ␤ mutator mutants. This screen takes advantage of a microbial genetics assay and the ability of rat pol ␤ to substitute for Escherichia coli DNA polymerase I in DNA replication in vivo. Using this screen, we identified 13 candidate pol ␤ mutator mutants. Three of the candidate mutator mutants were further characterized in vivo and shown to confer an increased spontaneous mutation frequency over that of wild-type pol ␤ to our bacterial strain. Purification and subsequent analysis of one of our putative mutator proteins, the pol ␤-14 protein, showed that it possesses intrinsic mutator activity in four different assays that measure the fidelity of DNA synthesis. Therefore, residue 265, which is altered in pol ␤-14 and another of our mutant proteins, pol ␤-166, is probably critical for accurate DNA synthesis by pol ␤. Thus, our genetic method of screening for pol ␤ mutator mutants is useful in identifying active mammalian DNA polymerase mutants that encode enzymes that catalyze DNA synthesis with altered fidelity compared with the wild-type pol ␤ enzyme.

DNA polymerase ␤ (pol ␤) is an ideal system for studying the role of its different amino acid residues in the fidelity of DNA synthesis. In this study, the T79S variant of pol ␤ was identified using an in vivo genetic screen. T79S is located in the N-terminal 8-kDa domain of pol ␤ and has no contact with either the DNA template or the incoming dNTP substrate. The T79S protein produced 8-fold more multiple mutations in the herpes simplex virus type 1-thymidine kinase assay than wild-type pol ␤. Surprisingly, T79S is a misincorporation mutator only when using a 3-recessed primer-template. In the presence of a single nucleotide-gapped DNA substrate, T79S displays an antimutator phenotype when catalyzing DNA synthesis opposite template C and has similar fidelity as wild type opposite templates A, G, or T. Threonine 79 is located directly between two helix-hairpinhelix motifs located within the 8-kDa and thumb domains of pol ␤. As the pol ␤ enzyme closes into its active form, the helix-hairpin-helix motifs appear to assist in the production and stabilization of a 90 o bend of the DNA. The function of the bent DNA is to present the templating base to the incoming nucleotide substrate. We propose that Thr-79 is part of a hydrogen bonding network within the helix-hairpin-helix motifs that is important for positioning the DNA within the active site. We suggest that alteration of Thr-79 to Ser disrupts this hydrogen bonding network and results in an enzyme that is unable to bend the DNA into the proper geometry for accurate DNA synthesis.1 has quickly become one of the best studied polymerases because the gene for the enzyme was cloned (1, 2). The availability of multiple crystal structures of human and rat pol ␤, including those of the enzyme complexed with both of its substrates and the metal cofactor, has aided the investigation of the structure-function relationships of this enzyme (3-8).pol ␤ is a 39-kDa protein with both nucleotidyltransferase and 5Ј-deoxyribose phosphodiesterase activities (9, 10). Evidence has been provided for a role for pol ␤ in both base excision repair and meiosis (11-13). There is no evidence that pol ␤ functions in replication of the mammalian genome, but pol ␤ has been shown to participate in DNA replication in Escherichia coli in the absence of DNA polymerase I (14). Mice that are completely deficient in pol ␤ die at 18 days post-conception due to massive apoptosis of post-mitotic neurons, suggesting that pol ␤ is essential for embryonic development (15, 16). The physiological DNA substrate for pol ␤ is believed to be a small gap because it has been shown that pol ␤ is processive on gaps of 6 bases or less and that the activity and fidelity of pol ␤ are highest on a 1-bp gap with a 5Ј-phosphate (17, 18).DNA polymerase ␤ has a modular organization with an 8-kDa N-terminal domain connected to the 31-kDa C-terminal domain by a protease-hypersensitive hinge region. The N-terminal 8-kDa domain was originally characterized as a singlestranded DNA binding domain (19,20). Subsequently, it was foun...

Purpose:To evaluate the potential of gene expression signatures to predict response to treatment in locally advanced cervical cancer treated with definitive chemotherapy and radiation. Experimental Design: Tissue biopsies were collected from patients participating in Radiation Therapy Oncology Group (RTOG) 0128, a phase II trial evaluating the benefit of celecoxib in addition to cisplatin chemotherapy and radiation for locally advanced cervical cancer. Gene expression profiling was done and signatures of pretreatment, mid-treatment (before the first implant), and ''changed''gene expression patterns between pre-and mid-treatment samples were determined. The ability of the gene signatures to predict local control versus local failure was evaluated. Two-group t test was done to identify the initial gene set separating these end points. Supervised classification methods were used to enrich the gene sets. The results were further validated by leave-one-out and 2-fold cross-validation. Results: Twenty-two patients had suitable material from pretreatment samples for analysis, and 13 paired pre-and mid-treatment samples were obtained. The changed gene expression signatures between the pre-and mid-treatment biopsies predicted response to treatment, separating patients with local failures from those who achieved local control with a seven-gene signature. The in-sample prediction rate, leave-one-out prediction rate, and 2-fold prediction rate are 100% for this seven-gene signature. This signature was enriched for cell cycle genes. Conclusions: Changed gene expression signatures during therapy in cervical cancer can predict outcome as measured by local control. After further validation, such findings could be applied to direct additional therapy for cervical cancer patients treated with chemotherapy and radiation.