Abstract
DNA polymerase β (Pol β) plays a key role in base excision repair (BER) by filling in small gaps that are generated after base adducts are excised from the DNA. Pol β is mutated in a large number of colorectal tumors, and these mutations may drive carcinogenesis. In the present study, we wished to determine whether the S229L somatic Pol β variant identified in a stage 3 colorectal tumor is a driver of carcinogenesis. We show that S229L does not possess any defects in binding to either DNA or nucleotides compared with the WT enzyme, but exhibits a significant loss of polymerization efficiency, largely due to an 8-fold decrease in the polymerization rate. S229L participates in BER, but due to its lower catalytic rate, does so more slowly than WT. Expression of S229L in mammalian cells induces the accumulation of BER intermediate substrates, chromosomal aberrations, and cellular transformation. Our results are consistent with the interpretation that S229L is a driver of carcinogenesis, likely as a consequence of its slow polymerization activity during BER in vivo.
Highlights
The POLB gene is mutated in 40% of human colorectal tumors
S229L Pol  Is a Slow Polymerase—In an initial screen of Pol  variants identified in our original colon tumor study [6], S229L exhibited a decreased rate of catalysis compared with Pol  WT in a primer extension assay [6]
S229L Pol  Binds DNA and dNTP with an Affinity Similar to WT, but Has a Defect in DNA Synthesis—To determine the biochemical mechanism underlying the slow rate of DNA synthesis by S229L Pol , we first examined the ability of the protein to bind to a 1-base pair gapped DNA substrate (45AG) (Table 1) by electrophoretic mobility shift assay (EMSA)
Summary
Results: The S229L variant is a slow DNA polymerase that leads to the accumulation of BER intermediates and induces cellular transformation. We wished to determine whether the S229L somatic Pol  variant identified in a stage 3 colorectal tumor is a driver of carcinogenesis. The mutations were not clustered in any specific region of the protein and were located in all four subdomains Some of these nonsynonymous mutations are likely a significant contributor to cancer progression because the mutations are selected for with many of the mutations being identified in late stage tumors, and there were no corresponding mutations identified in the normal matched tissues [6]. We sought to determine whether the S229L variant identified in a stage 3 colorectal tumor could drive carcinogenesis. Our results suggest that the S229L variant results in aberrant BER and that S229L can act as a driver of tumorigenesis
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