When the DNA polymerase that replicates the Escherichia coli chromosome, DNA polymerase III, makes an error, there are two primary defenses against mutation: proofreading by the ϵ subunit of the holoenzyme and mismatch repair. In proofreading-deficient strains, mismatch repair is partially saturated and the cell's response to DNA damage, the SOS response, may be partially induced. To investigate the nature of replication errors, we used mutation accumulation experiments and whole-genome sequencing to determine mutation rates and mutational spectra across the entire chromosome of strains deficient in proofreading, mismatch repair, and the SOS response. We report that a proofreading-deficient strain has a mutation rate 4000-fold greater than wild-type strains. While the SOS response may be induced in these cells, it does not contribute to the mutational load. Inactivating mismatch repair in a proofreading-deficient strain increases the mutation rate another 1.5-fold. DNA polymerase has a bias for converting G:C to A:T base pairs, but proofreading reduces the impact of these mutations, helping to maintain the genomic G:C content. These findings give an unprecedented view of how polymerase and error-correction pathways work together to maintain E. coli's low mutation rate of 1 per 1000 generations.