NGS-based analysis of base-substitution signatures created by yeast DNA polymerase eta and zeta on undamaged and abasic DNA templates in vitro

Translesion synthesis (TLS) is the mechanism in which DNA polymerases (TLS polymerases) bypass unrepaired template damage with high error rates. DNA polymerase eta and zeta (Pol eta and Pol zeta) are major TLS polymerases that are conserved from yeast to humans. In this study, we quantified frequencies of base-substitutions by yeast Pol eta and Pol zeta on undamaged and abasic templates in vitro. For accurate quantification, we used a next generation sequencing (NGS)-based method where DNA products were directly analyzed by parallel sequencing. On undamaged templates, Pol eta and Pol zeta showed distinct base-substitution profiles, and the substitution frequencies were differently influenced by the template sequence. The base-substitution frequencies were influenced mainly by the adjacent bases both upstream and downstream of the substitution sites. Thus we present the base-substitution signatures of these polymerases in a three-base format. On templates containing abasic sites, Pol eta created deletions at the lesion in more than 50% of the TLS products, but the formation of the deletions was suppressed by the presence of Pol zeta. Pol zeta and Pol eta cooperatively facilitated the TLS reaction over an abasic site in vitro, suggesting that these two polymerases can cooperate in efficient and high fidelity TLS.