Single-molecule studies reveal the dynamics of DNA repair and transcription-associated proteins

Intrinsically disordered regions (IDR) are protein segments that lack a defined tertiary structure. IDRs are enriched in eukaryotic chromatin-binding proteins, where they modulate protein-DNA and protein-protein interactions. In this thesis, I probe the function(s) of IDRs via two case studies: the yeast mismatch repair (MMR) protein Mlh1- Pms1 and transcription factors (TFs) derived from C. albicans, a pathogenic yeast. Using single-molecule DNA curtain assays, I demonstrate novel roles for IDRs in promoting facilitated diffusion of Mlh1-Pms1 on DNA. IDRs improve Mlh1-Pms1’s ability to bypass a single nucleosome and to navigate dense nucleosome arrays that resemble chromatin. Moreover, these IDRs are critical for the Mlh1-Pms1 ATPase activity and also for nicking of the DNA substrate. I propose that conformational changes in the Mlh1-Pms1 IDRs alter DNA interactions and the nucleolytic activity of neighboring structured domains. I also examine the dynamics of PCNA, another essential MMR factor, in the context of trinucleotide repeat (TNR) instability. I show that Replication Factor C preferentially loads PCNA onto (CAG)₁₃ structures. The (CAG)₁₃ repeat captures the loaded PCNA and prevents PCNA from diffusing. Lastly, I reveal a novel role for IDRs in DNA condensation by studying Efg1, a TF that regulates a cell-type switching network in C. albicans. Efg1 encodes a specific IDR of low complexity, referred to as the prion-like domain (PrLD). I show that the PrLD is critical for the DNA condensation and recruiting other PrLD-containing TFs, wherein nucleosomes regulate the TF-DNA dynamics. I propose a model where transcription factors become concentrated via phase separation and bring gene regulatory elements together to promote gene activation. Overall, this study provides mechanistic insights into the functions of IDRs in the dynamic behavior of DNA-binding proteinsBiochemistr