Competition Between Transcription Factors and Pol-δ During Okazaki Fragment Maturation Drives Site-Specific Mutagenesis

Abstract

Mutagenesis is a fundamental and complex biological process. Despite the remarkable fidelity of DNA replication, diverse mutational processes succeed at introducing new genomic variation, thereby providing a substrate for natural selection and genetic disorders. A thorough understanding of the nature of mutations, as well as their origins, is thus of high relevance to evolutionary biology, as well as mechanistic studies of human disease. During DNA replication, the lagging strand is synthesized as a set of discontinuous Okazaki fragments (OFs) initiated by the error-prone polymerase-α (Pol-α). During OF maturation, the higher-fidelity polymerase-δ (Pol-δ) displaces and re-synthesizes the Pol-α-created portions of OFs, removing mis-incorporated bases. However, transcription factors (TFs) that rapidly re-associate with DNA after replication fork passage can impede this displacement, potentially leading to the retention of error-prone Pol-α-synthesized DNA in the mature genome. Prior work established that three general regulatory factors (GRFs), Abf1, Reb1, and Rap1, strongly bias Okazaki fragment junction (OJ) positions, but whether this mutational mechanism extends broadly across TFs, and how specific TF-polymerase collisions influence mutagenesis at the nucleotide level, remains poorly understood. This study combines computational genomics and in vitro biochemistry in Saccharomyces cerevisiae to comprehensively characterize the relationship between TF binding and replication-associated mutagenesis. In the computational analysis, OJ frequencies and interspecies nucleotide substitution rates were mapped at the binding sites of 35 sequence-specific TFs. This genome-wide survey identified four GRFs whose binding sites exhibit a significant correlation between OJ accumulation and elevated substitution rates, extending prior observations to a broader set of factors and establishing the specificity of this phenomenon. To directly test the mechanistic basis of TF-mediated replication impediment, an in vitro strand displacement assay was developed using reconstituted yeast replication components and the TF Cbf1 as a model DNA-bound barrier. This assay was adapted for high-throughput measurement of strand displacement efficiency in the presence of Cbf1, enabling systematic evaluation of how specific nucleotide mismatches within the Cbf1 binding motif modulate the interplay between TF binding and Pol-δ processivity. The results demonstrate that Cbf1 binding impedes Pol-δ strand displacement synthesis, and that certain mismatches within the Cbf1 recognition sequence further reduce strand displacement efficiency in the presence of bound Cbf1. Importantly, these mismatches correspond to the nucleotide substitutions enriched at Cbf1 binding sites in the computational genomic analysis and are consistent with patterns reported in the literature. The convergence of the in vitro biochemical data with the genome-wide substitution patterns provides direct evidence that TF-mediated impediment of OF maturation contributes to site-specific mutagenesis at regulatory elements. These findings support a model in which the mutational cost of TF binding shapes the evolution of regulatory DNA, and they offer a mechanistic explanation for the enrichment of mutations at functionally important genomic sites.