Abstract The overall objective of this proposal is to answer two fundamental questions on DNA metabolism: 1) how DNA replication progresses timely and efficiently at hard-to-replicate sites; and 2) how the large nucleoprotein complexes that comprise telomeres, the end of linear chromosomes, are assembled and regulated. The first question emerges from our ongoing mechanistic studies on the conserved Pif1-family of helicases. Our work highlights the fundamental role that these helicases have in facilitating DNA replication at both secondary DNA structures and protein barriers. Going forward, we seek to address the following: what mechanisms couple the helicase activity to DNA synthesis by polymerases; how this synergy leads to removal of obstacles imparted by protein barriers encountered in the nucleus or in mitochondria; whether removal of obstacles is a general property of this class of helicases. The second question is novel and is based on our recent finding of new properties of both yeast and human telomere DNA binding proteins; namely, the ability of these proteins to interact with telomeric DNA repeats in alternative modes and to facilitate DNA condensation. Our goal is to test whether these properties play a role in establishing telomere length discrimination and accessibility. We tackle these problems by using biochemically reconstituted systems and employing a multipronged approach that integrates ensemble biochemical and biophysical techniques with single-molecule methods, thus providing unparalleled access to the behavior of individual molecules.