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Structural basis for topological regulation of Tn3 resolvase.

ABSTRACT: Site-specific DNA recombinases play a variety of biological roles, often related to the dissemination of antibiotic resistance, and are also useful synthetic biology tools. The simplest site-specific recombination systems will recombine any two cognate sites regardless of context. Other systems have evolved elaborate mechanisms, often sensing DNA topology, to ensure that only one of multiple possible recombination products is produced. The closely related resolvases from the Tn3 and γδ transposons have historically served as paradigms for the regulation of recombinase activity by DNA topology. However, despite many proposals, models of the multi-subunit protein-DNA complex (termed the synaptosome) that enforces this regulation have been unsatisfying due to a lack of experimental constraints and incomplete concordance with experimental data. Here, we present new structural and biochemical data that lead to a new, detailed model of the Tn3 synaptosome, and discuss how it harnesses DNA topology to regulate the enzymatic activity of the recombinase.

SUBMITTER: Montano SP

PROVIDER: S-EPMC9943657 | biostudies-literature | 2023 Feb

REPOSITORIES: biostudies-literature

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Structural basis for topological regulation of Tn3 resolvase.

Montaño Sherwin P SP Rowland Sally-J SJ Fuller James R JR Burke Mary E ME MacDonald Alasdair I AI Boocock Martin R MR Stark W Marshall WM Rice Phoebe A PA

Nucleic acids research 20230201 3

Site-specific DNA recombinases play a variety of biological roles, often related to the dissemination of antibiotic resistance, and are also useful synthetic biology tools. The simplest site-specific recombination systems will recombine any two cognate sites regardless of context. Other systems have evolved elaborate mechanisms, often sensing DNA topology, to ensure that only one of multiple possible recombination products is produced. The closely related resolvases from the Tn3 and γδ transposo ...[more]

PMID: 36100255

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Project description:Higher-order chromosomal organization for transcription regulation is poorly understood in eukaryotes. Using genome-wide Chromatin Interaction Analysis with Paired-End-Tag sequencing (ChIA-PET), we mapped long-range chromatin interactions associated with RNA polymerase II in human cells and uncovered widespread promoter-centered intragenic, extragenic, and intergenic interactions. These interactions further aggregated into higher-order clusters, wherein proximal and distal genes were engaged through promoter-promoter interactions. Most genes with promoter-promoter interactions were active and transcribed cooperatively, and some interacting promoters could influence each other implying combinatorial complexity of transcriptional controls. Comparative analyses of different cell lines showed that cell-specific chromatin interactions could provide structural frameworks for cell-specific transcription, and suggested significant enrichment of enhancer-promoter interactions for cell-specific functions. Furthermore, genetically-identified disease-associated noncoding elements were found to be spatially engaged with corresponding genes through long-range interactions. Overall, our study provides insights into transcription regulation by three-dimensional chromatin interactions for both housekeeping and cell-specific genes in human cells. RNA polymerase II (RNAPII) bound chromatin interactions were extracted with Chromatin Interaction Analysis with Paired-End Tag (ChIA-PET) sequencing, in order to study the transcription regulations with RNAPII-associated long-range chromatin interactions. Five cell lines, namely MCF7 (ATCC# HTB-22), K562 (ATCC# CCL-243), HCT116 (ATCC# CCL-247), HeLa (ATCC# CCL-2.2), and NB4 (Roussel and Lanotte, 2001) (provided by Dr. Sherman Weissman, Yale University), were grown under standard culture conditions and harvested at log phase. Harvested cells were cross-linked using 1% formaldehyde followed by neutralization with 0.2M glycine. Chromatin was isolated and subjected to ChIA-PET protocol as described in Fullwood et al (Fullwood et al: An oestrogen-receptor-alpha-bound human chromatin interactome. Nature 2009, 462(7269):58-64). The ChIA-PET sequence reads were processed and analyzed using ChIA-PET Tool (Li et al: ChIA-PET tool for comprehensive chromatin interaction analysis with paired-end tag sequencing. Genome Biol 2010, 11(2):R22).

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Structural basis for topological regulation of Tn3 resolvase.

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Structural basis for topological regulation of Tn3 resolvase.

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