The ability to express or deplete proteins in living cells is crucial for the study of biological processes. Viral vectors are often useful to deliver DNA constructs to cells that are difficult to transfect by other methods. Lentiviruses have the additional advantage of being able to integrate into the genomes of non-dividing mammalian cells. However, existing viral expression systems generally require different vector backbones for expression of cDNA, small hairpin RNA (shRNA) or microRNA (miRNA) and provide limited drug selection markers. Furthermore, viral backbones are often recombinogenic in bacteria, complicating the generation and maintenance of desired clones. Here, we describe a collection of 59 vectors that comprise an integrated system for constitutive or inducible expression of cDNAs, shRNAs or miRNAs, and use a wide variety of drug selection markers. These vectors are based on the Gateway technology (Invitrogen) whereby the cDNA, shRNA or miRNA of interest is cloned into an Entry vector and then recombined into a Destination vector that carries the chosen viral backbone and drug selection marker. This recombination reaction generates the desired product with >95% efficiency and greatly reduces the frequency of unwanted recombination in bacteria. We generated Destination vectors for the production of both retroviruses and lentiviruses. Further, we characterized each vector for its viral titer production as well as its efficiency in expressing or depleting proteins of interest. We also generated multiple types of vectors for the production of fusion proteins and confirmed expression of each. We demonstrated the utility of these vectors in a variety of functional studies. First, we show that the FKBP12 Destabilization Domain system can be used to either express or deplete the protein of interest in mitotically-arrested cells. Also, we generate primary fibroblasts that can be induced to senesce in the presence or absence of DNA damage. Finally, we determined that both isoforms of the AT-Rich Interacting Domain 4B (ARID4B) protein could induce G1 arrest when overexpressed. As new technologies emerge, the vectors in this collection can be easily modified and adapted without the need for extensive recloning.

Mutations in XPD helicase, required for nucleotide excision repair (NER) as part of the transcription/repair complex TFIIH, cause three distinct phenotypes: cancer-prone xeroderma pigmentosum (XP), or aging disorders Cockayne syndrome (CS), and trichothiodystrophy (TTD). To clarify molecular differences underlying these diseases, we determined crystal structures of the XPD catalytic core from Sulfolobus acidocaldarius and measured mutant enzyme activities. Substrate-binding grooves separate adjacent Rad51/RecA-like helicase domains (HD1, HD2) and an arch formed by 4FeS and Arch domains. XP mutations map along the HD1 ATP-binding edge and HD2 DNA-binding channel and impair helicase activity essential for NER. XP/CS mutations both impair helicase activity and likely affect HD2 functional movement. TTD mutants lose or retain helicase activity but map to sites in all four domains expected to cause framework defects impacting TFIIH integrity. These results provide a foundation for understanding disease consequences of mutations in XPD and related 4Fe-4S helicases including FancJ.

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.

Helicases must unwind DNA at the right place and time to maintain genomic integrity or gene expression. Biologically critical XPB and XPD helicases are key members of the human TFIIH complex; they anchor CAK kinase (cyclinH, MAT1, CDK7) to TFIIH and open DNA for transcription and for repair of duplex distorting damage by nucleotide excision repair (NER). NER is initiated by arrested RNA polymerase or damage recognition by XPC-RAD23B with or without DDB1/DDB2. XP helicases, named for their role in the extreme sun-mediated skin cancer predisposition xeroderma pigmentosum (XP), are then recruited to asymmetrically unwind dsDNA flanking the damage. XPB and XPD genetic defects can also cause premature aging with profound neurological defects without increased cancers: Cockayne syndrome (CS) and trichothiodystrophy (TTD). XP helicase patient phenotypes cannot be predicted from the mutation position along the linear gene sequence and adjacent mutations can cause different diseases. Here we consider the structural biology of DNA damage recognition by XPC-RAD23B, DDB1/DDB2, RNAPII, and ATL, and of helix unwinding by the XPB and XPD helicases plus the bacterial repair helicases UvrB and UvrD in complex with DNA. We then propose unified models for TFIIH assembly and roles in NER. Collective crystal structures with NMR and electron microscopy results reveal functional motifs, domains, and architectural elements that contribute to biological activities: damaged DNA binding, translocation, unwinding, and ATP driven changes plus TFIIH assembly and signaling. Coupled with mapping of patient mutations, these combined structural analyses provide a framework for integrating and unifying the rich biochemical and cellular information that has accumulated over forty years of study. This integration resolves puzzles regarding XP helicase functions and suggests that XP helicase positions and activities within TFIIH detect and verify damage, select the damaged strand for incision, and coordinate repair with transcription and cell cycle through CAK signaling.

Damaged bases in DNA are known to lead to errors in replication and transcription, compromising the integrity of the genome. We have proposed a model where repair proteins containing redoxactive [4Fe-4S] clusters utilize DNA charge transport (CT) as a first step in finding lesions. In this model, the population of sites to search is reduced by a localization of protein in the vicinity of lesions. Here, we examine this model using single-molecule atomic force microscopy (AFM). XPD, a 5′-3′ helicase involved in nucleotide excision repair, contains a [4Fe-4S] cluster and exhibits a DNAbound redox potential that is physiologically relevant. In AFM studies, we observe the redistribution of XPD onto kilobase DNA strands containing a single base mismatch, which is not a specific substrate for XPD but, like a lesion, inhibits CT. We further provide evidence for DNA-mediated signaling between XPD and Endonuclease III (EndoIII), a base excision repair glycosylase that also contains a [4Fe-4S] cluster. When XPD and EndoIII are mixed together, they coordinate in relocalizing onto the mismatched strand. However, when a CT-deficient mutant of either repair protein is combined with the CT-proficient repair partner, no relocalization occurs. These data not only indicate a general link between the ability of a repair protein to carry out DNA CT and its ability to redistribute onto DNA strands near lesions but also provide evidence for coordinated DNA CT between different repair proteins in their search for damage in the genome.DNA electron transfer | iron-sulfur clusters | oxidative damage

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.

DNA polymerase ⑀ (pol ⑀) has been implicated in DNA replication, DNA repair, and cell cycle control, but its precise roles are unclear. When the subcellular localization of human pol ⑀ was examined by indirect immunofluorescence, pol ⑀ appeared in discrete nuclear foci that colocalized with proliferating cell nuclear antigen (PCNA) foci and sites of DNA synthesis only late in S phase. Early in S phase, pol ⑀ foci were adjacent to PCNA foci. In contrast to PCNA foci that were only present in S phase, pol ⑀ foci were present throughout mitosis and the G 1 phase of cycling cells. It is hypothesized from these observations that pol ⑀ and PCNA have separate but associated functions early in S phase and that pol ⑀ participates with PCNA in DNA replication late in S phase.Although as many as 13 DNA polymerases are known to exist in eukaryotes, only 3 are thought to be involved in chromosomal replication; they are DNA polymerases (pol) 1 ␣, ␦, and ⑀. pol ␣ is clearly involved in the synthesis and extension of RNA primers during DNA replication initiation (reviewed in Ref. 1), whereas pol ␦ is responsible for elongation, as most clearly demonstrated in SV40 DNA replication (2, 3). Although pol ⑀ is not required for SV40 replication (4, 5), it does appear to have a role in chromosomal DNA replication (5-10).DNA pol ⑀ is well conserved in sequence and subunit composition among eukaryotes. Human pol ⑀ has four subunits, a large catalytic subunit, p261, and three associated subunits, p59, p12, and p17 (11,12). Likewise, Saccharomyces cerevisiae pol ⑀ has a catalytic subunit of 256 kDa, POL2, and has three associated subunits encoded by DPB2, DPB3, DPB4 (8, 13-15).Both catalytic subunits are divided into two domains linked by a protease-sensitive region, an N-terminal domain containing polymerase and exonuclease motifs and a 120-kDa C-terminal domain. The S. cerevisiae and human catalytic subunits are 39% identical overall and have 63% identity in the N-terminal domain (16). p59 and DPB2p have 26% overall identity (17), whereas p17 and p12 have 36.5 and 34% identity to Dpb4p and Dpb3p, respectively (12). Given the conservation of pol ⑀ sequence and subunit structure between yeast and humans, it is likely that their functions are also largely conserved.A role for pol ⑀ in chromosomal DNA replication has been established in higher eukaryotes and in yeast. The elongation step of DNA replication is impaired in Xenopus laevis egg extracts immunodepleted of pol ⑀ (6). Additionally, using a polymerase trap technique that tags a polymerase with its DNA product, Zlotkin et al. (5) find that pol ⑀ from monkey kidney cells associates with nascent chromosomal DNA but not nascent SV40 DNA. Likewise, a neutralizing antibody against human pol ⑀ inhibits chromosomal replication when microinjected into human fibroblasts but fails to inhibit SV40 DNA replication in vitro (7). In S. cerevisiae, disruptions of POL2 are lethal and result in the terminal dumbbell morphology characteristic of a defect in DNA replication (8). When shifted to the no...

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.