Characterizing the role of Elevated dNTP pools in sensitizing the replisome

PROJECT SUMMARY The fidelity of DNA replication is critical in ensuring the stability of the genome from cell division to the next. Accurate DNA synthesis by replicative DNA requires appropriate levels and ratios of deoxyribonucleotide triphosphates (dNTPs), the building blocks of DNA, and is enhanced by the polymerase proofreading exonuclease function. The mismatch repair (MMR) system functions as a spell-check for DNA replication, detecting and directing repair of replication errors that evade proofreading. When dNTP pools are dysregulated, it interferes with the normal functioning of the replisome. Elevated dNTP pools lead to an increased rate of nucleotide misincorporation, increase the rate of DNA replication and alter the number of replication origins that are activated. Altering the dNTP pools in different ways (elevated, skewed) leads to distinct mutation profiles. These misincorporation events are substrates for MMR, but as the mutation rates increase, MMR can become saturated. Thus, altered dNTPs can promote mutagenesis that can lead to cancer. At the same time, cancer cells have elevated dNTP pools to maintain rapid proliferation. This can, in turn, lead to further mutagenesis and promote the molecular evolution of the cancer, providing a selective advantage. In this proposal, we hypothesize that elevated dNTP pools fundamentally change the activity of the replisome, both in terms of its function inaccurately synthesizing DNA and its ability to respond to DNA lesions and blocks that it encounters during DNA replication. In parallel, we are committed to the development of a diverse biomedical workforce, able to conduct this type of research. We hypothesize that a supportive network of researchers will support the inclusion of underrepresented groups in the biomedical sciences. In AIM 1, we will take advantage of the altered mutation profiles in rnr1 backgrounds to define the relative contributions of MLH complexes to MMR, which remains unknown. This will reveal important mechanistic details of the MMR pathway, including potential interactions among the MLH complexes. We will also define the mechanisms underlying the synthetic lethality between rnr1Y295A and mmr∆, potentially revealing a role for MMR in cell cycle checkpoint activation in the context of altered dNTP pools. In AIM 2, we will determine the mechanisms underlying the different ways in which elevated and/or skewed dNTP pools compromise the stability and normal progression of the replisome. We will characterize differences in how DSBs at the fork are processed when dNTP pools are elevated. We will assess Okazaki fragment processing in different rnr1 backgrounds. And we will use unbiased chemicogenomic screens to reveal pathways that are important in supporting cell viability when rnr1 strains are exposed to different DNA damaging agents that compromise replication fork progression. In AIM 3, we will establish a set of supportive programs that will provide research opportunities for a wide range of students at different academic levels. We will establish a Community Education Laboratory in Life Sciences that will offer authentic research experiences to K-12 students in Buffalo who are not typically exposed to possibilities in the biomedical workforce.