Dataset Information

Structure solution of DNA-binding proteins and complexes with ARCIMBOLDO libraries.

ABSTRACT: Protein-DNA interactions play a major role in all aspects of genetic activity within an organism, such as transcription, packaging, rearrangement, replication and repair. The molecular detail of protein-DNA interactions can be best visualized through crystallography, and structures emphasizing insight into the principles of binding and base-sequence recognition are essential to understanding the subtleties of the underlying mechanisms. An increasing number of high-quality DNA-binding protein structure determinations have been witnessed despite the fact that the crystallographic particularities of nucleic acids tend to pose specific challenges to methods primarily developed for proteins. Crystallographic structure solution of protein-DNA complexes therefore remains a challenging area that is in need of optimized experimental and computational methods. The potential of the structure-solution program ARCIMBOLDO for the solution of protein-DNA complexes has therefore been assessed. The method is based on the combination of locating small, very accurate fragments using the program Phaser and density modification with the program SHELXE. Whereas for typical proteins main-chain ?-helices provide the ideal, almost ubiquitous, small fragments to start searches, in the case of DNA complexes the binding motifs and DNA double helix constitute suitable search fragments. The aim of this work is to provide an effective library of search fragments as well as to determine the optimal ARCIMBOLDO strategy for the solution of this class of structures.

SUBMITTER: Propper K

PROVIDER: S-EPMC4051508 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

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Structure solution of DNA-binding proteins and complexes with ARCIMBOLDO libraries.

Pröpper Kevin K Meindl Kathrin K Sammito Massimo M Dittrich Birger B Sheldrick George M GM Pohl Ehmke E Usón Isabel I

Acta crystallographica. Section D, Biological crystallography 20140530 Pt 6

Protein-DNA interactions play a major role in all aspects of genetic activity within an organism, such as transcription, packaging, rearrangement, replication and repair. The molecular detail of protein-DNA interactions can be best visualized through crystallography, and structures emphasizing insight into the principles of binding and base-sequence recognition are essential to understanding the subtleties of the underlying mechanisms. An increasing number of high-quality DNA-binding protein str ...[more]

PMID: 24914984

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Project description:BACKGROUND:As most ancient biological samples have low levels of endogenous DNA, it is advantageous to enrich for specific genomic regions prior to sequencing. One approach-in-solution capture-enrichment-retrieves sequences of interest and reduces the fraction of microbial DNA. In this work, we implement a capture-enrichment approach targeting informative regions of the Y chromosome in six human archaeological remains excavated in the Caribbean and dated between 200 and 3000 years BP. We compare the recovery rate of Y-chromosome capture (YCC) alone, whole-genome capture followed by YCC (WGC?+?YCC) versus non-enriched (pre-capture) libraries. RESULTS:The six samples show different levels of initial endogenous content, with very low (<?0.05%, 4 samples) or low (0.1-1.54%, 2 samples) percentages of sequenced reads mapping to the human genome. We recover 12-9549 times more targeted unique Y-chromosome sequences after capture, where 0.0-6.2% (WGC?+?YCC) and 0.0-23.5% (YCC) of the sequence reads were on-target, compared to 0.0-0.00003% pre-capture. In samples with endogenous DNA content greater than 0.1%, we found that WGC followed by YCC (WGC?+?YCC) yields lower enrichment due to the loss of complexity in consecutive capture experiments, whereas in samples with lower endogenous content, the libraries' initial low complexity leads to minor proportions of Y-chromosome reads. Finally, increasing recovery of informative sites enabled us to assign Y-chromosome haplogroups to some of the archeological remains and gain insights about their paternal lineages and origins. CONCLUSIONS:We present to our knowledge the first in-solution capture-enrichment method targeting the human Y-chromosome in aDNA sequencing libraries. YCC and WGC?+?YCC enrichments lead to an increase in the amount of Y-DNA sequences, as compared to libraries not enriched for the Y-chromosome. Our probe design effectively recovers regions of the Y-chromosome bearing phylogenetically informative sites, allowing us to identify paternal lineages with less sequencing than needed for pre-capture libraries. Finally, we recommend considering the endogenous content in the experimental design and avoiding consecutive rounds of capture, as clonality increases considerably with each round.

Dataset Information

Structure solution of DNA-binding proteins and complexes with ARCIMBOLDO libraries.

Publications

Structure solution of DNA-binding proteins and complexes with ARCIMBOLDO libraries.

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