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Visualizing DNA replication by single-molecule analysis of replicated DNA.

ABSTRACT: Single-molecule analysis of replicated DNA (SMARD) is a unique technique that enables visualization of DNA replication at specific genomic regions at single-molecule resolution. Here, we present a protocol for visualizing DNA replication by SMARD. We describe steps for pulse labeling DNA, followed by isolating and stretching of genomic DNA. We then detail the detection of the replication at chromosomal regions through immunostaining and fluorescence in situ hybridization. Using SMARD, we can visualize replication initiation, progression, termination, and fork stalling. For complete details on the use and execution of this protocol, please refer to Norio et al. (2001) and Gerhardt et al. (2014).¹^,².

SUBMITTER: Madireddy A

PROVIDER: S-EPMC10730367 | biostudies-literature | 2023 Dec

REPOSITORIES: biostudies-literature

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Visualizing DNA replication by single-molecule analysis of replicated DNA.

Madireddy Advaitha A Gerhardt Jeannine J

STAR protocols 20231203 4

Single-molecule analysis of replicated DNA (SMARD) is a unique technique that enables visualization of DNA replication at specific genomic regions at single-molecule resolution. Here, we present a protocol for visualizing DNA replication by SMARD. We describe steps for pulse labeling DNA, followed by isolating and stretching of genomic DNA. We then detail the detection of the replication at chromosomal regions through immunostaining and fluorescence in situ hybridization. Using SMARD, we can vis ...[more]

PMID: 38048218

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Project description:Kaposi's sarcoma associated herpesvirus (KSHV), an etiologic agent of Kaposi's sarcoma, Body Cavity Based Lymphoma and Multicentric Castleman's Disease, establishes lifelong latency in infected cells. The KSHV genome tethers to the host chromosome with the help of a latency associated nuclear antigen (LANA). Additionally, LANA supports replication of the latent origins within the terminal repeats by recruiting cellular factors. Our previous studies identified and characterized another latent origin, which supported the replication of plasmids ex-vivo without LANA expression in trans. Therefore identification of an additional origin site prompted us to analyze the entire KSHV genome for replication initiation sites using single molecule analysis of replicated DNA (SMARD). Our results showed that replication of DNA can initiate throughout the KSHV genome and the usage of these regions is not conserved in two different KSHV strains investigated. SMARD also showed that the utilization of multiple replication initiation sites occurs across large regions of the genome rather than a specified sequence. The replication origin of the terminal repeats showed only a slight preference for their usage indicating that LANA dependent origin at the terminal repeats (TR) plays only a limited role in genome duplication. Furthermore, we performed chromatin immunoprecipitation for ORC2 and MCM3, which are part of the pre-replication initiation complex to determine the genomic sites where these proteins accumulate, to provide further characterization of potential replication initiation sites on the KSHV genome. The ChIP data confirmed accumulation of these pre-RC proteins at multiple genomic sites in a cell cycle dependent manner. Our data also show that both the frequency and the sites of replication initiation vary within the two KSHV genomes studied here, suggesting that initiation of replication is likely to be affected by the genomic context rather than the DNA sequences.

Dataset Information

Visualizing DNA replication by single-molecule analysis of replicated DNA.

Publications

Visualizing DNA replication by single-molecule analysis of replicated DNA.

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