The rate of DNA mutation is known to fluctuate across the genome but the patterns of mutation rate variation and molecular causes are poorly defined. It is important to understand these patterns of mutation as they influence where deleterious mutations are likely to arise and how rapidly sequences are likely to accumulate change between species, a measure often used as a proxy for functional constraint. In this work I investigate the relationship between DNA replication and apparent mutation hotspots adjacent to transcription factor binding sites. In eukaryotes both DNA strands are replicated simultaneously, the leading strand as a continuous stretch and the lagging strand as a series of discrete Okazaki fragments that are subsequently ligated together. Some transcription factors are able to bind the DNA lagging strand during replication and act as a partial barrier to DNA polymerase, resulting in the accumulation of Okazaki fragment junctions adjacent to these sites. I find that mutation rate is correlated genome wide with Okazaki junction frequency, suggesting that Okazaki junction processing may be error-prone. We present a mechanistic hypothesis to explain this locally elevated mutation rate and propose a role for lagging strand replication and its error-prone Pol α tract retention in the formation of these hotspots. I test this hypothesis using Okazaki fragment sequencing data from the yeast Saccharomyces cerevisiae to identify peaks in Okazaki junctions. When these peaks are aligned and orientated, so that the direction of lagging strand replication is uniform, I find a peak in substitution rate immediately downstream of Okazaki junctions, precisely where Pol α tract retention is predicted to occur. Novel binding motifs are identified within the underlying DNA of these junctions that can be assigned to known strong and fast-binding transcription factors, previously implicated in the phasing of nucleosomes, such as Reb1. I show that mutation hotspots adjacent to transcription factor binding sites are a conserved feature of eukaryotic genomes. In the human genome I predict sites of preferential Pol α retention using DNase I hypersensitivity footprint data. We observe that those footprints predicted as germline-specific manifest an elevated mutation signature. I propose that the rapid binding of some transcription factors to DNA following replication is required for nucleosome positioning or other important functions, however this incurs a cost in terms of locally elevated mutation rate adjacent to and within the sequence specific binding site. As a consequence these binding sites are biologically important mutational hotspots whose functional significance has been systematically underestimated by standard measures of sequence constraint.

Indiana University-Purdue University Indianapolis (IUPUI) / Accurate DNA replication is vital for maintaining genomic stability. Consequently, the machinery required to drive this process is designed to ensure the meticulous maintenance of information. However, random misincorporation of errors reduce the fidelity of the DNA and lead to pre-mature aging and age-related disorders such as cancer and neurodegenerative diseases. Some of the incorporated errors are the result of the error prone DNA polymerase alpha (Pol α), which initiates synthesis on both the leading and lagging strand. Lagging strand synthesis acquires an increased number of polymerase α tracks because of the number of Okazaki fragments synthesized per round of the cell cycle (~50 million in mammalian cells). The accumulation of these errors invariably reduces the fidelity of the genome. Previous work has shown that these pol α tracks can be removed by two redundant pathways referred to as the short and long flap pathway. The long flap pathway utilizes a complex network of proteins to remove more of the misincorporated nucleotides than the short flap pathway which mediates the removal of shorter flaps. Lysine acetylation has been reported to modulate the function of the nucleases implicated in flap processing. The cleavage activity of the long flap pathway nuclease, Dna2, is stimulated by lysine acetylation while conversely lysine acetylation of the short flap pathway nuclease, FEN1, inhibits its activity. The major protein players implicated during Okazaki fragment processing (OFP) are known, however, the choice of the processing pathway and its regulation by lysine acetylation of its main players is yet unknown. This dissertation identifies three main findings: 1) Saccharomyces cerevisiae helicase, petite integration frequency (Pif1) is lysine acetylated by Esa1 and deacetylated by Rpd3 regulating its viability and biochemical properties including helicase, binding and ATPase activity ii) the single stranded DNA binding protein, human replication protein A (RPA) is modified by p300 and this modification stimulates its primary binding function and iii) lysine acetylated human RPA directs OFP towards the long flap pathway even for a subset of short flaps.