Polytene Chromosome Structure and Somatic Genome Instability

Spradling, Allan C.

doi:10.1101/sqb.2017.82.033670

Cited by 9 publications

(7 citation statements)

References 67 publications

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“…The URs that map outside of TADs are unusual in being very small, ranging in size from only 2.2 to 17 kb. A correlation between URs and TADs has been noted recently by others as well [ 28 , 29 ].…”

Section: Resultssupporting

confidence: 53%

Dynamic changes in ORC localization and replication fork progression during tissue differentiation

et al. 2018

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BackgroundGenomic regions repressed for DNA replication, resulting in either delayed replication in S phase or underreplication in polyploid cells, are thought to be controlled by inhibition of replication origin activation. Studies in Drosophila polytene cells, however, raised the possibility that impeding replication fork progression also plays a major role.ResultsWe exploited genomic regions underreplicated (URs) with tissue specificity in Drosophila polytene cells to analyze mechanisms of replication repression. By localizing the Origin Recognition Complex (ORC) in the genome of the larval fat body and comparing this to ORC binding in the salivary gland, we found that sites of ORC binding show extensive tissue specificity. In contrast, there are common domains nearly devoid of ORC in the salivary gland and fat body that also have reduced density of ORC binding sites in diploid cells. Strikingly, domains lacking ORC can still be replicated in some polytene tissues, showing absence of ORC and origins is insufficient to repress replication. Analysis of the width and location of the URs with respect to ORC position indicates that whether or not a genomic region lacking ORC is replicated is controlled by whether replication forks formed outside the region are inhibited.ConclusionsThese studies demonstrate that inhibition of replication fork progression can block replication across genomic regions that constitutively lack ORC. Replication fork progression can be inhibited in both tissue-specific and genome region-specific ways. Consequently, when evaluating sources of genome instability it is important to consider altered control of replication forks in response to differentiation.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-4992-3) contains supplementary material, which is available to authorized users.

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Section: Resultssupporting

confidence: 53%

Dynamic changes in ORC localization and replication fork progression during tissue differentiation

et al. 2018

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“…Importantly, TAMRA-polypyrrole-stained DNA exhibits a clear backbone compared with DAPI-stained DNA (Figure 5B ) in terms of fluorescence intensity contrast (Figure 5C and D ). Staining polytene chromosomal DNA with TAMRA-polypyrrole could demonstrate the band and interband patterns with a high-resolution, therefore, it is advantageous to study somatic genome instability ( 45 ), chromosomal organization of the genome, and protein immunolocalization ( 46 ). Accordingly, TAMRA-polypyrrole is a promising reagent for investigating large polytene structures as well as single large DNA molecules.…”

Section: Resultsmentioning

confidence: 99%

TAMRA-polypyrrole for A/T sequence visualization on DNA molecules

Lee

Kawamoto

Vaijayanthi

et al. 2018

Nucleic Acids Research

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Fluorophore-linked, sequence-specific DNA binding reagents can visualize sequence information on a large DNA molecule. In this paper, we synthesized newly designed TAMRA-linked polypyrrole to visualize adenine and thymine base pairs. A fluorescent image of the stained DNA molecule generates an intensity profile based on A/T frequency, revealing a characteristic sequence composition pattern. Computer-aided comparison of this intensity pattern with the genome sequence allowed us to determine the DNA sequence on a visualized DNA molecule from possible intensity profile pattern candidates for a given genome. Moreover, TAMRA-polypyrrole offers robust advantages for single DNA molecule detection: no fluorophore-mediated photocleavage and no structural deformation, since it exhibits a sequence-specific pattern alone without the use of intercalating dyes such as YOYO-1. Accordingly, we were able to identify genomic DNA fragments from Escherichia coli cells by aligning them to the genomic A/T frequency map based on TAMRA-polypyrrole-generated intensity profiles. Furthermore, we showed band and interband patterns of polytene chromosomal DNA stained with TAMRA-polypyrrole because it prefers to bind AT base pairs.

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“…Genome instability and mutagenesis associated with under-replicated regions of polytene chromosomes have been proposed as a contributing factor to the adaptability of Diptera to their environment [65]. Thus, understanding the molecular mechanisms of DSB repair in underreplicated regions can provide important insights into phenomena responsible for adaptation and evolution.…”

Section: Discussionmentioning

confidence: 99%

Arp2/3 and Unc45 maintain heterochromatin stability in Drosophila polytene chromosomes

Dialynas

Delabaere

Chiolo

2019

Preprint

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Repairing DNA double-strand breaks (DSBs) is particularly challenging in pericentromeric heterochromatin, where the abundance of repeated sequences exacerbates the risk of ectopic recombination. In Drosophila Kc cells, accurate homologous recombination (HR) repair of heterochromatic DSBs relies on the relocalization of repair sites to the nuclear periphery before Rad51 recruitment and strand invasion. This movement is driven by Arp2/3-dependent nuclear actin filaments and myosins' ability to walk along them. Conserved mechanisms enable the relocalization of heterochromatic DSBs in mouse cells, and their defects lead to massive ectopic recombination in heterochromatin and chromosome rearrangements. In Drosophila polytene chromosomes, extensive DNA movement is blocked by a stiff structure of chromosome bundles. Repair pathways in this context are poorly characterized, and whether heterochromatic DSBs relocalize in these cells is unknown. Here, we show that damage in heterochromatin results in relaxation of the heterochromatic chromocenter, consistent with a dynamic response in this structure. Arp2/3, the Arp2/3 activator Scar, and the myosin activator Unc45, are required for heterochromatin stability in polytene cells, suggesting that relocalization enables heterochromatin repair in this tissue. Together, these studies reveal critical roles for actin polymerization and myosin motors in heterochromatin repair and genome stability across different organisms and tissue types. Short Title: Nucleoskeleton functions in heterochromatic DNA repair. Impact StatementHeterochromatin relies on dedicated pathways for 'safe' recombinational repair. In mouse and fly cultured cells, DNA repair requires the movement of repair sites away from the heterochromatin 'domain' via nuclear actin filaments and myosins. Here, we explore the importance of these pathways in Drosophila salivary gland cells, which feature a stiff bundle of endoreduplicated polytene chromosomes. Repair pathways in polytene chromosomes are largely obscure and how nuclear dynamics operate in this context is unknown. We show that heterochromatin relaxes in response to damage, and relocalization pathways are necessary for repair and stability of heterochromatic sequences. This deepens our understanding of repair mechanisms in polytenes, revealing unexpected dynamics. It also provides a first understanding of nuclear dynamics responding to replication damage or rDNA breaks, providing a new understanding of the importance of the nucleoskeleton in genome stability. We expect these discoveries to shed light on tumorigenic processes, including therapy-induced cancer relapses.

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Polytene Chromosome Structure and Somatic Genome Instability

Cited by 9 publications

References 67 publications

Dynamic changes in ORC localization and replication fork progression during tissue differentiation

Dynamic changes in ORC localization and replication fork progression during tissue differentiation

TAMRA-polypyrrole for A/T sequence visualization on DNA molecules

Arp2/3 and Unc45 maintain heterochromatin stability in Drosophila polytene chromosomes

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