The DNA-binding domain of human papillomavirus type 18 E1. Crystal structure, dimerization, and DNA binding

High risk types of human papillomavirus, such as type 18 (HPV-18), cause cervical carcinoma, one of the most frequent causes of cancer death in women worldwide. DNA replication is one of the central processes in viral maintenance, and the machinery involved is an excellent target for the design of antiviral therapy. The papillomaviral DNA replication initiation protein E1 has origin recognition and ATP-dependent DNA melting and helicase activities, and it consists of a DNA-binding domain and an ATPase/helicase domain. While monomeric in solution, E1 binds DNA as a dimer. Dimerization occurs via an interaction of hydrophobic residues on a single alpha-helix of each monomer. Here we present the crystal structure of the monomeric HPV-18 E1 DNA-binding domain refined to 1.8-A resolution. The structure reveals that the dimerization helix is significantly different from that of bovine papillomavirus type 1 (BPV-1). However, we demonstrate that the analogous residues required for E1 dimerization in BPV-1 and the low risk HPV-11 are also required for HPV-18 E1. We also present evidence that the HPV-18 E1 DNA-binding domain does not share the same nucleotide and amino acid requirements for specific DNA recognition as BPV-1 and HPV-11 E1

A structural snapshot of base-pair opening in DNA

The response of double-helical DNA to torsional stress may be a driving force for many processes acting on DNA. The 1.55-A crystal structure of a duplex DNA oligonucleotide d(CCAGGCCTGG)(2) with an engineered crosslink in the minor groove between the central guanine bases depicts how the duplex can accommodate such torsional stress. We have captured in the same crystal two rather different conformational states. One duplex contains a strained crosslink that is stabilized by calcium ion binding in the major groove, directly opposite the crosslink. For the other duplex, the strain in the crosslink is relieved through partial rupture of a base pair and partial extrusion of a cytosine accompanied by helix bending. The sequence used is the target sequence for the HaeIII methylase, and this partially flipped cytosine is the same nucleotide targeted for extrusion by the enzyme. Molecular dynamics simulations of these structures show an increased mobility for the partially flipped-out cytosine

A triple helix stabilizes the 3' ends of long noncoding RNAs that lack poly(A) tails

The MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) locus is misregulated in many human cancers and produces an abundant long nuclear-retained noncoding RNA. Despite being transcribed by RNA polymerase II, the 3' end of MALAT1 is produced not by canonical cleavage/polyadenylation but instead by recognition and cleavage of a tRNA-like structure by RNase P. Mature MALAT1 thus lacks a poly(A) tail yet is expressed at a level higher than many protein-coding genes in vivo. Here we show that the 3' ends of MALAT1 and the MEN beta long noncoding RNAs are protected from 3'-5' exonucleases by highly conserved triple helical structures. Surprisingly, when these structures are placed downstream from an ORF, the transcript is efficiently translated in vivo despite the lack of a poly(A) tail. The triple helix therefore also functions as a translational enhancer, and mutations in this region separate this translation activity from simple effects on RNA stability or transport. We further found that a transcript ending in a triple helix is efficiently repressed by microRNAs in vivo, arguing against a major role for the poly(A) tail in microRNA-mediated silencing. These results provide new insights into how transcripts that lack poly(A) tails are stabilized and regulated and suggest that RNA triple-helical structures likely have key regulatory functions in vivo

siRNA carrying an (E)-vinylphosphonate moiety at the 5' end of the guide strand augments gene silencing by enhanced binding to human Argonaute-2

Efficient gene silencing by RNA interference (RNAi) in vivo requires the recognition and binding of the 5'- phosphate of the guide strand of an siRNA by the Argonaute protein. However, for exogenous siRNAs it is limited by the rapid removal of the 5'- phosphate of the guide strand by metabolic enzymes. Here, we have determined the crystal structure of human Argonaute-2 in complex with the metabolically stable 5'-(E)-vinylphosphonate (5'-E-VP) guide RNA at 2.5-A resolution. The structure demonstrates how the 5' binding site in the Mid domain of human Argonaute-2 is able to adjust the key residues in the 5'-nucleotide binding pocket to compensate for the change introduced by the modified nucleotide. This observation also explains improved binding affinity of the 5'-E-VP -modified siRNA to human Argonaute-2 in-vitro, as well as the enhanced silencing in the context of the trivalent N-acetylgalactosamine (GalNAc)-conjugated siRNA in mice relative to the un-modified siRNA

Engineering photocycle dynamics. Crystal structures and kinetics of three photoactive yellow protein hinge-bending mutants

Crystallographic and spectroscopic analyses of three hinge-bending mutants of the photoactive yellow protein are described. Previous studies have identified Gly(47) and Gly(51) as possible hinge points in the structure of the protein, allowing backbone segments around the chromophore to undergo large concerted motions. We have designed, crystallized, and solved the structures of three mutants: G47S, G51S, and G47S/G51S. The protein dynamics of these mutants are significantly affected. Transitions in the photocycle, measured with laser induced transient absorption spectroscopy, show rates up to 6-fold different from the wild type protein and show an additive effect in the double mutant. Compared with the native structure, no significant conformational differences were observed in the structures of the mutant proteins. We conclude that the structural and dynamic integrity of the region around these mutations is of crucial importance to the photocycle and suggest that the hinge-bending properties of Gly(51) may also play a role in PAS domain proteins where it is one of the few conserved residues

Dynamic look at DNA unwinding by a replicative helicase

A prerequisite for DNA replication is the unwinding of duplex DNA catalyzed by a replicative hexameric helicase. Despite a growing body of research, key elements of helicase mechanism remain under substantial debate. In particular, the number of DNA strands encircled by the helicase ring during unwinding and the ring orientation at the replication fork completely contrast in contemporary mechanistic models. Here we use single-molecule and ensemble assays to address these questions for the papillomavirus E1 helicase. We find that E1 unwinds DNA with a strand-exclusion mechanism, with the N-terminal side of the helicase ring facing the replication fork. We show that E1 generates strikingly heterogeneous unwinding patterns stemming from varying degrees of repetitive movements, which is modulated by the DNA-binding domain. Together, our studies reveal previously unrecognized dynamic facets of replicative helicase unwinding mechanisms

Structure of the active form of human Origin Recognition Complex and its ATPase motor module

Binding of the Origin Recognition Complex (ORC) to origins of replication marks the first step in the initiation of replication of the genome in all eukaryotic cells. Here, we report the structure of the active form of human ORC determined by X-ray crystallography and cryo-electron microscopy. The complex is composed of an ORC1/4/5 motor module lobe in an organization reminiscent of the DNA polymerase clamp loader complexes. A second lobe contains the ORC2/3 subunits. The complex is organized as a double-layered shallow corkscrew, with the AAA+ and AAA+-like domains forming one layer, and the winged-helix domains (WHDs) forming a top layer. CDC6 fits easily between ORC1 and ORC2, completing the ring and the DNA-binding channel, forming an additional ATP hydrolysis site. Analysis of the ATPase activity of the complex provides a basis for understanding ORC activity as well as molecular defects observed in Meier-Gorlin Syndrome mutations

CRL4-like Clr4 complex in Schizosaccharomyces pombe depends on an exposed surface of Dos1 for heterochromatin silencing

Repressive histone H3 lysine 9 methylation (H3K9me) and its recognition by HP1 proteins are necessary for pericentromeric heterochromatin formation. In Schizosaccharomyces pombe, H3K9me deposition depends on the RNAi pathway. Cryptic loci regulator 4 (Clr4), the only known H3K9 methyltransferase in this organism, is a subunit of the Clr4 methyltransferase complex (CLRC), whose composition is reminiscent of a CRL4 type cullin-RING ubiquitin ligase (CRL) including its cullin Cul4, the RING-box protein Pip1, the DNA damage binding protein 1 homolog Rik1, and the DCAF-like protein delocalization of Swi6 1 (Dos1). Dos2 and Stc1 have been proposed to be part of the complex but do not bear similarity to canonical ubiquitin ligase components. CLRC is an active E3 ligase in vitro, and this activity is necessary for heterochromatin assembly in vivo. The similarity between CLRC and the CRLs suggests that the WD repeat protein Dos1 will act to mediate target recognition and substrate specificity for CLRC. Here, we present a pairwise interaction screen that confirms a CRL4-like subunit arrangement and further identifies Dos2 as a central component of the complex and recruiter of Stc1. We determined the crystal structure of the Dos1 WD repeat domain, revealing an eight-bladed beta-propeller fold. Functional mapping of the putative target-binding surface of Dos1 identifies key residues required for heterochromatic silencing, consistent with Dos1's role as the specificity factor for the E3 ubiquitin ligase

Rapid generation of drug-resistance alleles at endogenous loci using CRISPR-Cas9 indel mutagenesis

Genetic alterations conferring resistance to the effects of chemical inhibitors are valuable tools for validating on-target effects in cells. Unfortunately, for many therapeutic targets such alleles are not available. To address this issue, we evaluated whether CRISPR-Cas9-mediated insertion/deletion (indel) mutagenesis can produce drug-resistance alleles at endogenous loci. This method takes advantage of the heterogeneous in-frame alleles produced following Cas9-mediated DNA cleavage, which we show can generate rare alleles that confer resistance to the growth-arrest caused by chemical inhibitors. We used this approach to identify novel resistance alleles of two lysine methyltransferases, DOT1L and EZH2, which are each essential for the growth of MLL-fusion leukemia cells. We biochemically characterized the DOT1L mutation, showing that it is significantly more active than the wild-type enzyme. These findings validate the on-target anti-leukemia activities of existing DOT1L and EZH2 inhibitors and reveal a simple method for deriving drug-resistance alleles for novel targets, which may have utility during early stages of drug development

NADP regulates the yeast GAL induction system

Transcriptional regulation of the galactose-metabolizing genes in Saccharomyces cerevisiae depends on three core proteins: Gal4p, the transcriptional activator that binds to upstream activating DNA sequences (UAS(GAL)); Gal80p, a repressor that binds to the carboxyl terminus of Gal4p and inhibits transcription; and Gal3p, a cytoplasmic transducer that, upon binding galactose and adenosine 5'-triphosphate, relieves Gal80p repression. The current model of induction relies on Gal3p sequestering Gal80p in the cytoplasm. However, the rapid induction of this system implies that there is a missing factor. Our structure of Gal80p in complex with a peptide from the carboxyl-terminal activation domain of Gal4p reveals the existence of a dinucleotide that mediates the interaction between the two. Biochemical and in vivo experiments suggests that nicotinamide adenine dinucleotide phosphate (NADP) plays a key role in the initial induction event