Gap-filling DNA synthesis by HeLa DNA polymerase alpha in an in vitro base excision DNA repair scheme.


The ability of HeLa DNA polymerase alpha to utilize gapped PM2 DNAs for synthesis in a model base excision DNA repair scheme was examined. Partially depurinated PM2 DNA was incised on the 5' side of apurinic sites with HeLa apurinic/apyrimidinic endonuclease II, then the baseless sugar was removed and gaps of defined mean lengths were introduced at these sites by exonucleolytic digestion with HeLa DNase V. Gaps smaller than approximately 15 nucleotides did not serve as efficient primer-templates for DNA polymerase alpha. Gaps with mean lengths of 20-63 nucleotides did support limited DNA synthesis, but such synthesis terminated after the gap was reduced to roughly 15 nucleotides. These products were not substrates for Escherichia coli DNA ligase. In contrast, HeLa DNA polymerase beta utilize as primer-templates all of the gapped DNA substrates tested though it acted more efficiently with the smaller gaps. Moreover, the beta-polymerase was capable of filling these gaps to completion. In the case of the gaps that remained after partial closure by DNA polymerase alpha, DNA polymerase beta incorporated roughly 15 nucleotides and formed a product which was a substrate for DNA ligase. These results suggest that in vivo DNA repair pathways that involve a gap-filling DNA synthesis reaction might utilize DNA polymerase alpha only for larger gaps.




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