Fe-S clusters are partners in the origin of life that predate cells, acetyl-CoA metabolism, DNA, and the RNA world. The double helix solved the mystery of DNA replication by base pairing for accurate copying. Yet, for genome stability necessary to life, the double helix has equally important implications for damage repair. Here we examine striking advances that uncover Fe-S cluster roles both in copying the genetic sequence by DNA polymerases and in crucial repair processes for genome maintenance, as mutational defects cause cancer and degenerative disease. Moreover, we examine an exciting, controversial role for Fe-S clusters in a third element required for life – the long-range coordination and regulation of replication and repair events. By their ability to delocalize electrons over both Fe and S centers, Fe-S clusters have unbeatable features for protein conformational control and charge transfer via double-stranded DNA that may fundamentally transform our understanding of life, replication, and repair.

Using DNA-modified electrodes, we show DNA-mediated signaling by XPD, a helicase that contains a [4Fe-4S] cluster and is critical for nucleotide excision repair and transcription. The DNA-mediated redox signal resembles that of base excision repair proteins, with a DNA-bound redox potential of ~80 mV versus NHE. Significantly, this signal increases with ATP hydrolysis. Moreover, the redox signal is substrate-dependent, reports on the DNA conformational changes associated with enzymatic function, and may reflect a general biological role for DNA charge transport.

Recombinant expression of large, multi-protein complexes is essential and often rate-limiting for determining structural, biophysical, and biochemical properties of DNA repair, replication, transcription, and other key cellular processes. Baculovirus infected insect cell expression systems are especially well-suited for producing large, human proteins recombinantly and multi-gene baculovirus systems have facilitated studies of multi-protein complexes. In this chapter, we describe a multi-gene baculovirus system called MacroBac that uses a Biobricks-type assembly method based on restriction and ligation (Series 11) or ligation independent cloning (LIC) (Series 438). MacroBac cloning and assembly is efficient and equally well-suited for either single subcloning reactions or high-throughput cloning using 96-well plates and liquid handling robotics. MacroBac vectors are polypromoter with each gene flanked by a strong polyhedrin promoter and an SV40 poly(A) termination signal that minimize gene order expression level effects seen in many polycistronic assemblies. Large assemblies are robustly achievable, and we have successfully assembled as many as 10 genes into a single MacroBac vector. Importantly, we have observed significant increases in expression levels and quality of large, multi-protein complexes using a single, multi-gene, polypromoter virus rather than co-infection with multiple, single-gene viruses. Given the importance of characterizing functional complexes, we believe that MacroBac provides a critical enabling technology that may change the way that structural, biophysical, and biochemical research is done.

SummaryArchaea employ the archaellum, a type IV pilus-like nanomachine, for swimming motility. In the crenarchaeon Sulfolobus acidocaldarius, the archaellum consists of seven proteins: FlaB/X/G/F/H/I/J. FlaF is conserved and essential for archaellum assembly but no FlaF structures exist. Here, we truncated the FlaF N terminus and solved 1.5-Å and 1.65-Å resolution crystal structures of this monotopic membrane protein. Structures revealed an N-terminal α-helix and an eight-strand β-sandwich, immunoglobulin-like fold with striking similarity to S-layer proteins. Crystal structures, X-ray scattering, and mutational analyses suggest dimer assembly is needed for in vivo function. The sole cell envelope component of S. acidocaldarius is a paracrystalline S-layer, and FlaF specifically bound to S-layer protein, suggesting that its interaction domain is located in the pseudoperiplasm with its N-terminal helix in the membrane. From these data, FlaF may act as the previously unknown archaellum stator protein that anchors the rotating archaellum to the archaeal cell envelope.