Analysis of the impact of a uracil DNA glycosylase attenuated in AP-DNA binding in maintenance of the genomic integrity in Escherichia coli

Abstract

Uracil DNA glycosylase (Ung) initiates the uracil excision repair pathway. We have earlier characterized the Y66W and Y66H mutants of Ung and shown that they are compromised by approximately 7- and approximately 170-fold, respectively in their uracil excision activities. In this study, fluorescence anisotropy measurements show that compared with the wild-type, the Y66W protein is moderately compromised and attenuated in binding to AP-DNA. Allelic exchange of ung in Escherichia coli with ung::kan, ungY66H:amp or ungY66W:amp alleles showed approximately 5-, approximately 3.0- and approximately 2.0-fold, respectively increase in mutation frequencies. Analysis of mutations in the rifampicin resistance determining region of rpoB revealed that the Y66W allele resulted in an increase in A to G (or T to C) mutations. However, the increase in A to G mutations was mitigated upon expression of wild-type Ung from a plasmid borne gene. Biochemical and computational analyses showed that the Y66W mutant maintains strict specificity for uracil excision from DNA. Interestingly, a strain deficient in AP-endonucleases also showed an increase in A to G mutations. We discuss these findings in the context of a proposal that the residency of DNA glycosylase(s) onto the AP-sites they generate shields them until recruitment of AP-endonucleases for further repair.

Figure 1.

Generation of fluorescein-labled SSap9 oligomer and its binding to the Ung proteins. (A) Scheme of uracil excision from F-SSU9 (a) to generate F-SSap9 (b). Cleavage of F-SSap9 (b) in the presence of alkali, or alkali and heat results in the formation of (c) and (d). Of these, (a), (b) and (c) are detectable on the gel shown in (B). (B) Analysis of uracil excision from F-SSU9 with Ung proteins. The DNA oligomer was incubated with Ung proteins (5 µM); aliquots were taken out and analyzed on 15% polyacrylamide–8 M urea gels before and after treatment with alkali and alkali and heat. Lane 1, untreated F-SSU9; Lanes 2, 5 and 8, F-SSU9 treated with wild-type, Y66W and Y66H Ung; lanes 3, 6 and 9, same as in lanes 2, 5 and 8, respectively after treatment with alkali; lanes 4, 7 and 10, same as in lanes 3, 6, and 9 after treatment with alkali and heat. (C) Determination of the specificity of F-SSap9 binding to the Ung. Complexes of Ung proteins with F-SSap9 (see ‘Materials and Methods’ section) were competed with increasing amounts of Ugi. Anisotropies, relative to the starting values of the respective complexes of F-SSap9 with Ung proteins (taken as 100%, in the absence of Ugi) were plotted against Ugi concentration. (D) Fluorescence anisotropy measurements using a double-stranded AP-DNA. The F-DSAU9 (1 μM) was mixed with 5 μM to 200 μM Ung proteins (wild-type, filled diamond; Y66W, filled triangle; and Y66H, filled circle) and the flluorescence anisotropy was measured in Jasco FP-777. Data points were fitted to binding equation (see ‘Materials and Methods’ section). The K_D values shown are the averages from two experiments ±SEM.

Figure 2.

Characterization of E. coli MG1655 strains. (A) Genomic organization of E. coli MG1655 strains harboring various ung alleles. (B) Immunoblot analysis of the cell-free extracts using anti-Ung and anti-RRF antibodies. Cell-free extracts prepared from E. coli MG1655 strain (lane 1), or its derivatives harboring amp^R linked wild-type (ung:amp, lane 2), Y66H (ungY66H:amp, lane 3), Y66W (ungY66H:amp, lane 4) and ung- (ung::kan, lane 5) alleles.

Figure 3.

In vitro excision of [³H] thymine from DNA. [³H] thymine containing DNA was incubated with the various Ung proteins (or their complexes with Ugi) for the indicated times. The thymine release was analyzed by chromatography on PEI thin layer plates, calculated as percentage release [(free thymine)/(free thymine + DNA substrate left)]*100 from three different experiments, and plotted as histograms. Error bars indicate standard deviation.

Figure 4.

Genomic degradation assays. (A) The genomic DNA (∼1.5 µg) from E. coli (wild-type for ung) was mixed with buffer alone (Buffer) or 500 ng of wild-type EcoUng (Ung), Y66W EcoUng (Y66W), Y147A mutant of human UNG (HsY147) in the absence (−) or the presence (+) of Ugi, incubated at 37°C for 3 h, treated with NaOH and heat, and analyzed by electrophoresis on agaraose gel and recorded. (B) Same as (A) except that the genomic DNA was from dut⁻ung⁻ strain of E. coli and the amount of Ung proteins used was 200 ng, and the reactions were done for 15 min.