Česlovas Venclovas

A modified definition of Sov, a segment‐based measure for protein secondary structure prediction assessment

We present a measure for the evaluation of secondary structure prediction methods that is based on secondary structure segments rather than individual residues. The algorithm is an extension of the segment overlap measure Sov, originally defined by Rost et al. (J Mol Biol 1994;235:13–26). The new definition of Sov corrects the normalization procedure and improves Sovs ability to discriminate between similar and dissimilar segment distributions. The method has been comprehensively tested during the second Critical Assessment of Techniques for Protein Structure Prediction (CASP2). Here, we describe the underlying concepts, modifications to the original definition, and their significance. Proteins 1999;34:220–223. Published 1999 Wiley‐Liss, Inc.

Structure-based predictions of Rad1, Rad9, Hus1 and Rad17 participation in sliding clamp and clamp-loading complexes

The repair of damaged DNA is coupled to the completion of DNA replication by several cell cycle checkpoint proteins, including, for example, in fission yeast Rad1(Sp), Hus1(Sp), Rad9(Sp) and Rad17(Sp). We have found that these four proteins are conserved with protein sequences throughout eukaryotic evolution. Using computational techniques, including fold recognition, comparative modeling and generalized sequence profiles, we have made high confidence structure predictions for the each of the Rad1, Hus1 and Rad9 protein families (Rad17(Sc), Mec3(Sc) and Ddc1(Sc) in budding yeast, respectively). Each of these families was found to share a common protein fold with that of PCNA, the sliding clamp protein that tethers DNA polymerase to its template. We used previously reported genetic and biochemical data for these proteins from yeast and human cells to predict a heterotrimeric PCNA-like ring structure for the functional Rad1/Rad9/Hus1 complex and to determine their exact order within it. In addition, for each individual protein family, contact regions with neighbors within the PCNA-like ring were identified. Based on a molecular model for Rad17(Sp), we concluded that members of this family, similar to the subunits of the RFC clamp-loading complex, are capable of coupling ATP binding with conformational changes required to load a sliding clamp onto DNA. This model substantiates previous findings regarding the behavior of Rad17 family proteins upon DNA damage and within the RFC complex of clamp-loading proteins.

Processing and Analysis of CASP3 Protein Structure Predictions

Livermore Prediction Center provides basic infrastructure for the CASP (Critical Assessment of Structure Prediction) experiments, including prediction processing and verification servers, a system of prediction evaluation tools, and interactive numerical and graphical displays. Here we outline the essentials of our approach, with discussion of the superposition procedures, definitions of basic measures, and descriptions of new methods developed to analyze predictions. Our primary focus is on the evaluation of threedimensional models and secondary structure predictions. To put the results of the three prediction experiments held to date on the same footing, the latest CASP3 evaluation criteria were retrospectively applied to both CASP1 and CASP2 predictions. Finally, we give an overview of our website (http://PredictionCenter.llnl.gov), which makes the target structures, predictions, and the evaluation system accessible to the community. Proteins Suppl 1999;3:22–29. Published 1999 Wiley‐Liss, Inc.

Progress over the first decade of CASP experiments.

CASP has now completed a decade of monitoring the state of the art in protein structure prediction. The quality of structure models produced in the latest experiment, CASP6, has been compared with that in earlier CASPs. Significant although modest progress has again been made in the fold recognition regime, and cumulatively, progress in this area is impressive. Models of previously unknown folds again appear to have modestly improved, and several mixed α/β structures have been modeled in a topologically correct manner. Progress remains hard to detect in high sequence identity comparative modeling, but server performance in this area has moved forward. Proteins 2005;61:225–236.

A Sliding Clamp Model for the Rad1 Family of Cell Cycle Checkpoint Proteins

We explored the possibility that the Rad1 family members have more distant homology with other proteins of Rad1 family were embedded into the Rad1 Mm sequence using BLOCKs, and this cobbled sequence was used as the probe in a PSI-BLAST search. An initial weak We have identified an unexpected structural similarity match was amplified after several iterations, and PCNA between six members of the Rad1 cell cycle checkpoint (S. cerevisiae, and other species) was retrieved as a family and the DNA sliding clamp protein PCNA. Like candidate homolog with nearly end-to-end sequence its prokaryotic structural homolog, the ␤ subunit of DNA alignment. However, PCNA and the probe sequence polymerase III, PCNA facilitates genome replication by were only 15% identical, making it uncertain whether encircling the DNA helix and tethering DNA polymerase these protein families are truly related. to its substrate. Our Rad1 model predicts that eukaryotic We therefore sought independent verification of this cells contain a second PCNA-like structure, one that hypothesis using the methods of fold recognition and may be critical to the mechanisms coupling DNA repair comparative modeling. Using an empirically derived fit-and DNA synthesis to a mitotic checkpoint. ness function, fold recognition (threading) evaluates the The Rad1 family of proteins, including Rec1 of Usti-compatibility of a new sequence with templates in the lago maydis and its distant relatives Rad1 sp (S. pombe) library of known folds. Threading was performed for and Rad17 sc (S. cerevisiae), function in both DNA repair each of the six homologs separately. In each case, only and cell cycle control (Lydall and Weinert, 1995; Onel the PCNA fold was always among the top ranking hits. Sequence comparisons reveal established false positive threshold (Fischer and Eisen-conserved blocks of amino acids between these six berg, 1996). proteins, but overall there is less than 30% identity be-The hypothesis was further explored by building and tween any two sequences. Similarity to other known evaluating an all atom model of Rad1 Mm. Using the PSI-proteins is not obvious, and a common biochemical BLAST alignment as a guide, Rad1 Mm amino acid side chains were placed on the peptide backbone of the function remains unclear. Rad1 homologs include those from mouse (Mm_Rad1), fruit fly (Dm_Rad1), smut fungus (Um_Rec1), and fission yeast (Sp_Rad1). PCNA sequences are from budding yeast (Sc_PCNA), malaria parasite (Pf_PCNA), bac-ulovirus (Ac_PCNA), and human (Hs_PCNA). Rad1 family sequence alignment with PCNA proteins relied on Rad1 Mm modeling …

Processing and evaluation of predictions in CASP4

The Livermore Prediction Center conducted the target collection and prediction submission processes for Critical Assessment of Protein Structure Prediction (CASP4) and Critical Assessment of Fully Automated Structure Prediction Methods (CAFASP2). We have also evaluated all the submitted predictions using criteria and methods developed during the course of three previous CASP experiments and preparation for CASP4. We present an overview of the implemented system. Particular attention is paid to newly developed evaluation techniques and data presentation schemes. With the rapid increase in CASP participation and in the number of submitted predictions, special emphasis is placed on methods allowing reliable pre‐classification of submissions and on techniques useful in automated evaluation of predictions. We also present an overview of our website, including target structures, predictions, and their evaluations ( http://predictioncenter.llnl.gov). Proteins 2001;Suppl 5:13–21.

Programmable rna shredding by the type iii-a crispr-cas system of streptococcus thermophilus

Immunity against viruses and plasmids provided by CRISPR-Cas systems relies on a ribonucleoprotein effector complex that triggers the degradation of invasive nucleic acids (NA). Effector complexes of type I (Cascade) and II (Cas9-dual RNA) target foreign DNA. Intriguingly, the genetic evidence suggests that the type III-A Csm complex targets DNA, whereas biochemical data show that the type III-B Cmr complex cleaves RNA. Here we aimed to investigate NA specificity and mechanism of CRISPR interference for the Streptococcus thermophilus Csm (III-A) complex (StCsm). When expressed in Escherichia coli, two complexes of different stoichiometry copurified with 40 and 72 nt crRNA species, respectively. Both complexes targeted RNA and generated multiple cuts at 6 nt intervals. The Csm3 protein, present in multiple copies in both Csm complexes, acts as endoribonuclease. In the heterologous E. coli host, StCsm restricts MS2 RNA phage in a Csm3 nuclease-dependent manner. Thus, our results demonstrate that the type III-A StCsm complex guided by crRNA targets RNA and not DNA.

Comparison of performance in successive CASP experiments

As the number of completed CASP (Critical Assessment of Protein Structure Prediction) experiments grows, so does the need for stable, standard methods for comparing performance in successive experiments. It is critical to develop methods for determining the areas in which there is progress and in which areas are static. We have added an analysis of the CASP4 results to that previously published for CASPs 1, 2, and 3. We again use a unified difficulty scale to permit comparison of performance as a function of target difficulty in the different CASPs. The scale is used to compare performance in aligning target sequences to a structural template. There was a clear improvement in alignment quality between CASP1 (1994) and CASP2 (1996). No change is apparent between CASP2 and CASP3 (1998). There is a small barely detectable improvement between CASP3 and the latest experiment (CASP4, 2000). Alignment remains the major source of error in all models based on less than about 30% sequence identity. Comparison of performance in the new fold modeling regime is complicated by issues in devising an objective target difficulty scale. We have found limited numerical support for significant progress between CASP3 and CASP4 in this area. More subjectively, most observers are convinced that there has been substantial progress. Progress is dominated by a single group. Proteins 2001;Suppl 5:163–170.

Comparative modeling in CASP6 using consensus approach to template selection, sequence‐structure alignment, and structure assessment

Along with over 150 other groups we have tested our template‐based protein structure prediction approach by submitting models for 30 target proteins to the sixth round of the Critical Assessment of Protein Structure Prediction Methods (CASP6, http://predictioncenter.org). Most of our modeled proteins fall into the comparative or homology modeling (CM) category, and some are fold recognition (FR) targets. The key feature of our structure prediction strategy in CASP6 was an attempt to optimally select structural templates and to make accurate sequence–structure alignments. Template selection was based mainly on consensus results of multiple sequence searches. Likewise, the consensus of multiple alignment variants (or lack of it) was used to initially delineate reliable and unreliable alignment regions. Structure evaluation approaches were then used to identify the correct sequence–structure mapping. Our results suggest that in many cases use of multiple templates is advantageous. Selecting correct alignments even within the context of a three‐dimensional structure remains a challenge. Together with more effective energy evaluation methods the simultaneous relaxation/refinement of a “frozen” backbone inherited from the template is likely needed to see a clear progress in tackling this problem. Our analysis also suggests that human input has little to contribute to automatic methods in modeling high homology targets. On the other hand, human expertise can be very valuable in modeling distantly related proteins and critical in cases of unexpected evolutionary changes in protein structure. Proteins 2005;Suppl 7:99–105.

Essential roles for imuA′- and imuB-encoded accessory factors in DnaE2-dependent mutagenesis in Mycobacterium tuberculosis

In Mycobacterium tuberculosis (Mtb), damage-induced mutagenesis is dependent on the C-family DNA polymerase, DnaE2. Included with dnaE2 in the Mtb SOS regulon is a putative operon comprising Rv3395c, which encodes a protein of unknown function restricted primarily to actinomycetes, and Rv3394c, which is predicted to encode a Y-family DNA polymerase. These genes were previously identified as components of an imuA-imuB-dnaE2–type mutagenic cassette widespread among bacterial genomes. Here, we confirm that Rv3395c (designated imuA′) and Rv3394c (imuB) are individually essential for induced mutagenesis and damage tolerance. Yeast two-hybrid analyses indicate that ImuB interacts with both ImuA′ and DnaE2, as well as with the β-clamp. Moreover, disruption of the ImuB-β clamp interaction significantly reduces induced mutagenesis and damage tolerance, phenocopying imuA′, imuB, and dnaE2 gene deletion mutants. Despite retaining structural features characteristic of Y-family members, ImuB homologs lack conserved active-site amino acids required for polymerase activity. In contrast, replacement of DnaE2 catalytic residues reproduces the dnaE2 gene deletion phenotype, strongly implying a direct role for the α-subunit in mutagenic lesion bypass. These data implicate differential protein interactions in specialist polymerase function and identify the split imuA′-imuB/dnaE2 cassette as a compelling target for compounds designed to limit mutagenesis in a pathogen increasingly associated with drug resistance.