Disordered proteins regulate stress- & damage-dependent DNA binding

Abstract

Three-dimensional structure has been used as a starting point to characterize protein function since the advent of X-ray crystallography in the 1950s. However, not all proteins bear stable tertiary or even secondary structures. Referred to as intrinsically disordered protein regions, or IDRs, how then do we relate these structures (or lack thereof) to their biochemical function? The electrostatically charged and, counterintuitively, selectively inflexible nature of many IDRs point to a key macromolecular interaction partner with similar physical properties--DNA. In this dissertation, we explore the functions of three different IDRs: the C-terminus of E. coli single-stranded binding protein (SSB), the C-terminus of X. laevis XRCC4-like factor (XLF), and the N-terminus of the human estrogen receptor α (ESR1). In each of these cases, these IDRs are crucial for regulating the access of other factors or domains to DNA during times of cellular stress and DNA damage. For SSB, we use single-molecule imaging in live cells to show that the stable exposure of its C-terminus during replication fork stalling facilitates the recruitment of stall-resolution factors. From site-directed mutagenesis and ensemble biochemical end-joining assays, we demonstrate that the C-terminal extension of double-strand break repair factor XLF enables ligation machinery to fluidly access breaks while maintaining a bridge between DNA ends. Finally, preliminary NMR and DNA binding data show that phosphorylations to the ESR1 N-terminus induced by oxidative stress pathways during cancer progression reduce its propensity to form any discernible structure as well as its surprisingly sequence-specific DNA affinity. In each of these cases, the ability of the IDR to assume an extended conformation in an activated state allows for the procession of key cellular processes, including replication progression, DNA repair, and the transcription of genes in response to DNA damage.