No abstract

Proliferating cell nuclear antigen (PCNA) plays critical roles in many aspects of DNA replication and replication-associated processes, including translesion synthesis, error-free damage bypass, break-induced replication, mismatch repair, and chromatin assembly. Since its discovery, our view of PCNA has evolved from a replication accessory factor to the hub protein in a large protein-protein interaction network that organizes and orchestrates many of the key events at the replication fork. We begin this review article with an overview of the structure and function of PCNA. We discuss the ways its many interacting partners bind and how these interactions are regulated by post-translational modifications such as ubiquitylation and sumoylation. We then explore the many roles of PCNA in normal DNA replication and in replication-coupled DNA damage tolerance and repair processes. We conclude by considering how PCNA can interact physically with so many binding partners to carry out its numerous roles. We propose that there is a large, dynamic network of linked PCNA molecules at and around the replication fork. This network would serve to increase the local concentration of all the proteins necessary for DNA replication and replication-associated processes and to regulate their various activities.

Though the importance of high-resolution structure and dynamics of membrane proteins has been well recognized, optimizing sample conditions to retain the native-like folding and function of membrane proteins for Nuclear Magnetic Resonance (NMR) or X-ray measurements has been a major challenge. While bicelles have been shown to stabilize the function of membrane proteins and are increasingly utilized as model membranes, the loss of their magnetic-alignment at low temperatures makes them unsuitable to study heat-sensitive membrane proteins like cytochrome-P450 and protein-protein complexes. In this study, we report temperature resistant bicelles that can magnetically-align for a broad range of temperatures and demonstrate their advantages in the structural studies of full-length microsomal cytochrome-P450 and cytochrome-b5 by solid-state NMR spectroscopy. Our results reveal that the N-terminal region of rabbit cytochromeP4502B4, that is usually cleaved off to obtain crystal structures, is helical and has a transmembrane orientation with ~17° tilt from the lipid bilayer normal.

DNA damage leads to genome instability by interfering with DNA replication. Cells possess several damage bypass pathways that mitigate the effects of DNA damage during replication. These pathways include translesion synthesis and template switching. These pathways are regulated largely through post-translational modifications of proliferating cell nuclear antigen (PCNA), an essential replication accessory factor. Mono-ubiquitylation of PCNA promotes translesion synthesis, and K63-linked poly-ubiquitylation promotes template switching. This article will discuss the mechanisms of how these post-translational modifications of PCNA control these bypass pathways from a structural and biochemical perspective. We will focus on the structure and function of the E3 ubiquitin ligases Rad18 and Rad5 that facilitate the mono-ubiquitylation and poly-ubiquitylation of PCNA, respectively. We conclude by reviewing alternative ideas about how these post-translational modifications of PCNA regulate the assembly of the multi-protein complexes that promote damage bypass pathways.

Proteins and protein complexes with high conformational flexibility participate in a wide range of biological processes. These processes include genome maintenance, gene expression, signal transduction, cell cycle regulation, and many others. Gaining a structural understanding of conformationally flexible proteins and protein complexes is arguably the greatest problem facing structural biologists today. Over the last decade, some progress has been made toward understanding the conformational flexibility of such systems using hybrid approaches. One particularly fruitful strategy has been the combination of small-angle X-ray scattering (SAXS) and molecular simulations. In this article, we provide a brief overview of SAXS and molecular simulations and then discuss two general approaches for combining SAXS data and molecular simulations: minimal ensemble approaches and full ensemble approaches. In minimal ensemble approaches, one selects a minimal ensemble of structures from the simulations that best fit the SAXS data. In full ensemble approaches, one validates a full ensemble of structures from the simulations using SAXS data. We argue that full ensemble models are more realistic than minimal ensemble searches models and that full ensemble approaches should be used wherever possible.