The Mechanism and Regulation of Bacteriophage DNA Packaging Motors

Many double-stranded DNA viruses use a packaging motor during maturation to recognize and transport genetic material into the capsid. In terminase motors, the TerS complex recognizes DNA, while the TerL motor packages the DNA into the capsid shell. Although there are several models for DNA recognition and translocation, how the motor components assemble and power DNA translocation is unknown. Using the thermophilic P74-26 bacteriophage model system, we discover that TerL uses a trans-activated ATP hydrolysis mechanism. Additionally, we identify critical residues for TerL ATP hydrolysis and DNA binding. With a combination of x-ray crystallography, SAXS, and molecular docking, we build a structural model for TerL pentamer assembly. Apo and ATP analog-bound TerL ATPase domain crystal structures show ligand-dependent conformational changes, which we propose power DNA translocation. Together, we assimilate these findings to build models for both motor assembly and DNA translocation. Additionally, with the P76-26 system, we identify the TerS protein as gp83. I find that P74-26 TerS is a nonameric ring that stimulates TerL ATPase activity while inhibiting TerL nuclease activity. Using cryoEM, I solve 3.8 Å and 4.8 Å resolution symmetric and asymmetric reconstructions of the TerS ring. I observe in P74-26 TerS, the conserved C-terminal beta-barrel is absent, and instead the region is flexible or unstructured. Furthermore, the helix-turn-helix motifs of P74-26 TerS are positioned differently than those of known TerS structures, suggesting P74-26 uses an alternative mechanism to recognize DNA

A thermophilic phage uses a small terminase protein with a fixed helix-turn-helix geometry [preprint]

Tailed bacteriophage use a DNA packaging motor to encapsulate their genome during viral particle assembly. The small terminase (TerS) component acts as a molecular matchmaker by recognizing the viral genome as well as the main motor component, the large terminase (TerL). How TerS binds DNA and the TerL protein remains unclear. Here, we identify the TerS protein of the thermophilic bacteriophage P74-26. TerSP76-26 oligomerizes into a nonamer that binds DNA, stimulates TerL ATPase activity, and inhibits TerL nuclease activity. Our cryo-EM structure shows that TerSP76-26 forms a ring with a wide central pore and radially arrayed helix-turn-helix (HTH) domains. These HTH domains, which are thought to bind DNA by wrapping the helix around the ring, are rigidly held in an orientation distinct from that seen in other TerS proteins. This rigid arrangement of the putative DNA binding domain imposes strong constraints on how TerSP76-26 can bind DNA. Finally, the TerSP76-26 structure lacks the conserved C-terminal β-barrel domain used by other TerS proteins for binding TerL, suggesting that a well-ordered C-terminal β-barrel domain is not necessary for TerS to carry out its function as a matchmaker

The large terminase DNA packaging motor grips DNA with its ATPase domain for cleavage by the flexible nuclease domain

Many viruses use a powerful terminase motor to pump their genome inside an empty procapsid shell during virus maturation. The large terminase (TerL) protein contains both enzymatic activities necessary for packaging in such viruses: the adenosine triphosphatase (ATPase) that powers DNA translocation and an endonuclease that cleaves the concatemeric genome at both initiation and completion of genome packaging. However, how TerL binds DNA during translocation and cleavage remains mysterious. Here we investigate DNA binding and cleavage using TerL from the thermophilic phage P74-26. We report the structure of the P74-26 TerL nuclease domain, which allows us to model DNA binding in the nuclease active site. We screened a large panel of TerL variants for defects in binding and DNA cleavage, revealing that the ATPase domain is the primary site for DNA binding, and is required for nuclease activity. The nuclease domain is dispensable for DNA binding but residues lining the active site guide DNA for cleavage. Kinetic analysis of DNA cleavage suggests flexible tethering of the nuclease domains during DNA cleavage. We propose that interactions with the procapsid during DNA translocation conformationally restrict the nuclease domain, inhibiting cleavage; TerL release from the capsid upon completion of packaging unlocks the nuclease domains to cleave DNA

Assembly of human C-terminal binding protein (CtBP) into tetramers

C-terminal binding protein 1 (CtBP1) and CtBP2 are transcriptional coregulators that repress numerous cellular processes, such as apoptosis, by binding transcription factors and recruiting chromatin-remodeling enzymes to gene promoters. The NAD(H)-linked oligomerization of human CtBP is coupled to its co-transcriptional activity, which is implicated in cancer progression. However, the biologically relevant level of CtBP assembly has not been firmly established; nor has the stereochemical arrangement of the subunits above that of a dimer. Here, multi-angle light scattering (MALS) data established the NAD(+)- and NADH-dependent assembly of CtBP1 and CtBP2 into tetramers. An examination of subunit interactions within CtBP1 and CtBP2 crystal lattices revealed that both share a very similar tetrameric arrangement resulting from assembly of two dimeric pairs, with specific interactions probably being sensitive to NAD(H) binding. Creating a series of mutants of both CtBP1 and CtBP2, we tested the hypothesis that the crystallographically observed interdimer pairing stabilizes the solution tetramer. MALS data confirmed that these mutants disrupt both CtBP1 and CtBP2 tetramers, with the dimer generally remaining intact, providing the first stereochemical models for tetrameric assemblies of CtBP1 and CtBP2. The crystal structure of a subtle destabilizing mutant suggested that small structural perturbations of the hinge region linking the substrate- and NAD-binding domains are sufficient to weaken the CtBP1 tetramer. These results strongly suggest that the tetramer is important in CtBP function, and the series of CtBP mutants reported here can be used to investigate the physiological role of the tetramer

Resistance to therapy in BRCA2 mutant cells due to loss of the nucleosome remodeling factor CHD4

Hereditary cancers derive from gene defects that often compromise DNA repair. Thus, BRCA-associated cancers are sensitive to DNA-damaging agents such as cisplatin. The efficacy of cisplatin is limited, however, by the development of resistance. One cisplatin resistance mechanism is restoration of homologous recombination (HR), which can result from BRCA reversion mutations. However, in BRCA2 mutant cancers, cisplatin resistance can occur independently of restored HR by a mechanism that remains unknown. Here we performed a genome-wide shRNA screen and found that loss of the nucleosome remodeling factor CHD4 confers cisplatin resistance. Restoration of cisplatin resistance is independent of HR but correlates with restored cell cycle progression, reduced chromosomal aberrations, and enhanced DNA damage tolerance. Suggesting clinical relevance, cisplatin-resistant clones lacking genetic reversion of BRCA2 show de novo loss of CHD4 expression in vitro. Moreover, BRCA2 mutant ovarian cancers with reduced CHD4 expression significantly correlate with shorter progression-free survival and shorter overall survival. Collectively, our findings indicate that CHD4 modulates therapeutic response in BRCA2 mutant cancer cells

Phase II Efficacy and Pharmacogenomic Study of Selumetinib (AZD6244; ARRY-142886) in Iodine-131 Refractory Papillary Thyroid Carcinoma with or without Follicular Elements

A multicenter, open-label, phase II trial was conducted to evaluate the efficacy, safety, and tolerability of selumetinib in iodine-refractory papillary thyroid cancer (IRPTC)

Understanding Attitudes toward Energy Security: Results of a Cross-National Survey

Energy security is embedded in a complex system encompassing factors that constitute the social environment in which individuals are immersed. Everything from education, to access to resources to policy and cultural values of particular places affects perceptions and experiences of energy security. This article examines the types of energy security challenges that nations face and characterizes the policy responses that are often used to address these challenges. Drawing from a survey of energy consumers in ten countries, we conduct a cross-national comparison of energy security attitudes and analyze each country’s corresponding energy resources, consumption characteristics and energy policies. Through multivariate regression analysis and case studies we find that socio-demographic and regional characteristics affect attitudes towards energy security. Specifically, a strong relationship exists between level of reliance on oil imports and level of concern for a variety of energy security characteristics including availability, affordability and equity. Our results also reaffirm the importance of gender and age in shaping perceptions of security. Level of development, reliance on oil and strong energy efficiency policies also affect individuals’ sense of energy security. In sum, we find that energy security is a highly context-dependent condition that is best understood from a nuanced and multi-dimensional perspective