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