RecBCD enzyme: mechanistic insights from mutants of a complex helicase-nuclease.

SUMMARYRecBCD enzyme is a multi-functional protein that initiates the major pathway of homologous genetic recombination and DNA double-strand break repair in Escherichia coli. It is also required for high cell viability and aids proper DNA replication. This 330-kDa, three-subunit enzyme is one of the fastest, most processive helicases known and contains a potent nuclease controlled by Chi sites, hotspots of recombination, in DNA. RecBCD undergoes major changes in activity and conformation when, during DNA unwinding, it encounters Chi (5'-GCTGGTGG-3') and nicks DNA nearby. Here, we discuss the multitude of mutations in each subunit that affect one or another activity of RecBCD and its control by Chi. These mutants have given deep insights into how the multiple activities of this complex enzyme are coordinated and how it acts in living cells. Similar studies could help reveal how other complex enzymes are controlled by inter-subunit interactions and conformational changes.

The Origin Recognition Complex: From Origin Selection to Replication Licensing in Yeast and Humans.

Understanding human DNA replication through the study of yeast has been an extremely fruitful journey. The minichromosome maintenance (MCM) 2-7 genes that encode the catalytic core of the eukaryotic replisome were initially identified through forward yeast genetics. The origin recognition complexes (ORC) that load the MCM hexamers at replication origins were purified from yeast extracts. We have reached an age where high-resolution cryoEM structures of yeast and human replication complexes can be compared side-by-side. Their similarities and differences are converging as alternative strategies that may deviate in detail but are shared by both species.

Structures of Class I and Class II Transcription Complexes Reveal the Molecular Basis of RamA-Dependent Transcription Activation.

Transcription activator RamA is linked to multidrug resistance of Klebsiella pneumoniae through controlling genes that encode efflux pumps (acrA) and porin-regulating antisense RNA (micF). In bacteria, σ70 , together with activators, controls the majority of genes by recruiting RNA polymerase (RNAP) to the promoter regions. RNAP and σ70 form a holoenzyme that recognizes -35 and -10 promoter DNA consensus sites. Many activators bind upstream from the holoenzyme and can be broadly divided into two classes. RamA acts as a class I activator on acrA and class II activator on micF, respectively. The authors present biochemical and structural data on RamA in complex with RNAP-σ70 at the two promoters and the data reveal the molecular basis for how RamA assembles and interacts with core RNAP and activates transcription that contributes to antibiotic resistance. Further, comparing with CAP/TAP complexes reveals common and activator-specific features in activator binding and uncovers distinct roles of the two C-terminal domains of RNAP α subunit.

Cryo-EM structures reveal the molecular basis of receptor-initiated coxsackievirus uncoating.

Enterovirus uncoating receptors bind at the surface depression ("canyon") that encircles each capsid vertex causing the release of a host-derived lipid called "pocket factor" that is buried in a hydrophobic pocket formed by the major viral capsid protein, VP1. Coxsackievirus and adenovirus receptor (CAR) is a universal uncoating receptor of group B coxsackieviruses (CVB). Here, we present five high-resolution cryoEM structures of CVB representing different stages of virus infection. Structural comparisons show that the CAR penetrates deeper into the canyon than other uncoating receptors, leading to a cascade of events: collapse of the VP1 hydrophobic pocket, high-efficiency release of the pocket factor and viral uncoating and genome release under neutral pH, as compared with low pH. Furthermore, we identified a potent therapeutic antibody that can neutralize viral infection by interfering with virion-CAR interactions, destabilizing the capsid and inducing virion disruption. Together, these results define the structural basis of CVB cell entry and antibody neutralization.