Abstract Single stranded DNA (ssDNA) has been demonstrated to by extremely vulnerable to DNA damage. Cancers often carry long stretches of clustered mutations that likely arose due to damage of ssDNA. While, various studies have demonstrated that certain mutagens preferentially damage ssDNA, the mechanisms that alter mutation specificity due to damage in ssDNA and the pathways that prevent mutagenesis at ssDNA are unknown. The overarching goal of this proposal is to specifically identify the roles of DNA damage checkpoint proteins, translesion polymerases, ssDNA-specific glycosylases and ssDNA binding proteins in altering the mutation patterns obtained upon ssDNA-specific damage. My laboratory is in a unique position to advance this scientific front based on my strong track record in DNA damage and repair, assembled team of collaborators, and multidisciplinary approach. My expertise in using highly sensitive yeast reporter systems, human cell culture techniques and the use of bioinformatics tools to probe large data sets and to analyze next generation sequencing data allow us to develop our research program to understand the pathways modulating ssDNA mutagenesis in yeast and human cells. Previously, I have demonstrated that alkylating agents and acetaldehyde have an ssDNA-specific mutation signature in yeast and in cancers. These mutation signatures provide us with a highly sensitive tool to determine how changes in various DNA repair, damage bypass and damage sensing pathways alter mutagenesis by ssDNA-specific mutagens. Here, we propose to determine 1) How cell cycle dependent translesion polymerase expression alters mutation signatures in ssDNA; 2) The role of ssDNA binding Replication Protein A complex in protecting ssDNA from exogenous damage; 3) The role of DNA damage checkpoint activation in modulating the mutation signatures associated with ssDNA damage; 4) Which DNA glycosylases function on ssDNA and alter the mutation signatures due to ssDNA damage; and 5) What are the mutagenic outcomes when translesion polymerases are unable to bypass ssDNA damage. This set of research projects will address a key gap in knowledge in understanding the mechanisms that alter the hypermutability of ssDNA in cells. Our work will enable us to identify and develop better cancer preventative measures for individuals who are prone to increased genome instability and ssDNA formation in their cells.